CN106551925A - Tung oil tree kind shell refines lignans extract, preparation method and applications - Google Patents
Tung oil tree kind shell refines lignans extract, preparation method and applications Download PDFInfo
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Abstract
The invention belongs to pharmaceutical technology field, there is provided a kind of tung oil tree kind shell refined lignans extract, preparation method and applications, the lignans extract is mainly by different phytolaccanine A (isoamerican A), phytolaccanine B (american B), (±) 3, 3 '-dinor- rosin spirit ((±) 3, 3 '-bisdemethylpinoresinol), 7S, 8R-americanin D are constituted, the extract in vivo and in vitro can be antitumor, inducing apoptosis of tumour cell, treatment nerve degenerative diseases, can be used to prevent and treat neuroinflamation, the development and application of cancer drug.
Description
Technical field
The invention belongs to pharmaceutical technology field, and in particular to lignanoid, preparation method and the application for preparing prevention and treatment neuroinflammatory conditions, cancer drug are refined in tung oil tree (Vernicia fordii).
Background technology
Tung oil tree (Vernicia fordii), also known as aleurites fordii, tung tree, tung tree, seeds of a tung oil tree tree, light paulownia, is Euphorbiaceae tung oil tree platymiscium.Tung oil tree is cold in nature, sweet, micro-pungent, poisonous.Its root, leaf, seed can be used as medicine.The whole world totally 3 kinds, are distributed in east Asia area, have 2 kinds in China, and main product is saved in Hunan, Sichuan, Guizhou etc..
Described in the dictionary of medicinal plant that tung oil tree is most published early in Science and Technology of Shanghai publishing house in the recent period for 1986, its leaf, root, seed are respectively provided with antiinflammatory action[1].Nineteen ninety, document report《Fujian traditional herbal medicine》,《Vegetation is just square》Recording tungoiltree juvenile leaf has the effect of subdhing swelling and detoxicating, can treat carbuncle swells, and erysipelas, ulcer on the shank, pernio, mange are scalded, dysentery, enteritis[2]。
Research in the last few years to the pharmacological action of tung oil tree extract or wherein isolated compound is very few.It is documented that, in early days in addition to the report that tung oil has treating cancer, remaining mainly acts as compound using treating various burns with other Chinese medicines one, scalds, rheumatoid arthritis, rachitis etc..At present, to the anti-neuroinflamation of lignanoids in tung oil tree and antitumor activity in terms of not yet report.
The content of the invention
It is an object of the invention to provide a kind of tung oil tree kind shell refined lignans extract, preparation method and applications, the lignans extract has anti-inflammatory, suppresses the effect of growth of tumour cell, can be used to prevent and treat neuroinflamation, the development and application of cancer drug.
The lignans extract that the present invention is extracted in specifically providing a kind of kind shell from tung oil tree, it is characterized in that, mainly by different phytolaccanine A (isoamerican A), phytolaccanine B (american B), (±) 3,3 '-dinor- rosin spirit ((±) 3,3 '-bisdemethylpinoresinol), 7S, 8R-americanin D is constituted.
Lignans extract of the present invention, it is characterised in that：In the lignans extract, the percentage by weight of each compound is：Different phytolaccanine A >=62%, phytolaccanine B >=5%, (±) 3,3 '-dinor- rosin spirit >=12%, 7S, 8R-americanin D >=6%, total content≤100% of four kinds of main compounds.
Present invention also offers the preparation method of the lignans extract, it is characterised in that comprise the steps：
(1) tung oil tree kind shell, is dried, after crushing, uses solvent extraction, recovered under reduced pressure extract to obtain crude extract；
(2), by the process of said extracted thing Jing nonpolar macroporous adsorption resins, gradient elution being carried out with solvent, collecting and combining the eluent containing lignanoid, reduced pressure concentration obtains tung oil tree kind shell total lignan；
Or use petroleum ether or hexamethylene, dichloromethane or chloroform, ethyl acetate, extracting n-butyl alcohol, reduced pressure concentration extract to obtain total lignan successively said extracted liquid；
(3), the total lignan Jing polyamide column chromatographies obtained by step (2) are processed, and carry out gradient elution with solvent, collect and combine the eluent containing lignanoid, and reduced pressure concentration is dried, obtains refined lignanoid；
Or by the process of tung oil tree total lignan Jing silica gel column chromatographies, carry out gradient elution with organic solvent, collect and combine the eluent containing tung oil tree lignanoid, reduced pressure concentration, dry must refine tung oil tree lignanoid.
Wherein, in step (1), Extraction solvent is ethanol that volumetric concentration is 60%～100% or methyl alcohol, and extraction time is 1～3 time, and the volume (L) of medicinal material weight (kg) and Extraction solvent is than being 1:5～1:15, preferably 1:6～1:12.
In step (2), the mobile phase of macroporous adsorbent resin chromatography is methyl alcohol and water mixed solvent or ethanol and water mixed solvent, and in mixed solvent, the volume ratio of methyl alcohol or second alcohol and water is 1：9～8：2, preferably 25：75～50：50.
The number of times that extract is extracted successively in step (2) is 1-3 time, and extractant is 1 with the volume ratio of extract:1～3:1.
In step (3), polyamide column chromatography mobile phase is methyl alcohol and water mixed solvent or ethanol and water mixed solvent, and in mixed solvent, the volume ratio of methyl alcohol or second alcohol and water is 1：9～8：2, preferably 25：75～7：3.
In step (3), silica gel column chromatography mobile phase is dichloromethane and methyl alcohol, chloroform and methanol mixed solvent, and in mixed solvent, the volume ratio of dichloromethane or chloroform and methyl alcohol is 100：1～2：1, preferably 100:2～100:10.
Lignans extract of the present invention has anti-inflammatory, suppresses growth of tumour cell effect, can be applicable to prepare treatment neuroinflammatory conditions medicine and cancer therapy drug, or in terms of preparing the medicine with cancer cell specific induction of apoptosis effect.
The present invention is with the neural microglia of LPS inductions as model, external S180 anti-tumor experiments model, and the anti-inflammatory of the tung oil tree Zhong Ke lignanoids to preparing, active anticancer are evaluated.As a result show, tung oil tree Zhong Ke lignanoids can significantly inhibit the BV2 Activated Microglias of LPS inductions, significantly inhibit 9 kinds of lung cancer cell line (A549 of people, H226, H292, H460, H1792, SW1573, CALU-1, H1299, H358), different degrees of inhibitory activity is shown to human liver cancer cell HepG2, gastric carcinoma cells HGC-27 and chronic myeloid leukemia cell K562.Therefore, the tung oil tree Zhong Ke lignanoids for preparing in the present invention can be used to prevent and treat neuroinflamation, the development and application of cancer therapy drug.
Specific embodiment
The following examples will be further described to the present invention, but not thereby limiting the invention.
Embodiment 1
(1) tung oil tree kind shell 1kg is dried, with 100% methyl alcohol heating and refluxing extraction 2 times, the volume (L) of medicinal material weight (kg) and Extraction solvent is than for 1:10, recovered under reduced pressure extract obtains crude extract；
(2) extract Jing nonpolar macroporous adsorption resins obtained by step (1) are processed, with methyl alcohol and aqueous solvent：1:9,3:7,5:5,7:3,8:2 carry out gradient elution, collect 3:7,5:5,7:The eluent of 3 three flow points, reduced pressure concentration obtain tung oil tree Zhong Kecu lignanoids；
(3) tung oil tree Zhong Kecu lignanoid Jing polyamide column chromatographies obtained by step (2) are processed, with methyl alcohol and water mixed solvent：1:9,3:7,5:5,7:3,8:2 carry out gradient elution, collect and combine containing 3:7,5:5,7:3 three flow point eluents, reduced pressure concentration are dried, obtain refined tung oil tree total lignan, wherein different phytolaccanine A contents are 68.3%, and phytolaccanine B content is 5.5%, (±) 3,3 '-dinor- rosin alcohol content is 14.2%, 7S, and 8R-americanin D contents are 8.0%.
(4) gained tung oil tree kind shell total lignan in step (3), Jing silica gel column chromatographies are separated, with dichloromethane, methanol mixed solvent gradient elution, 4 main components of total lignan are prepared into, Jing spectral datas are analyzed and identified as different phytolaccanine A, phytolaccanine B, (±) 3,3 '-dinor- rosin spirit, 7S, 8R-americanin D.The Structural Identification data of compound are following (table 1-4)
Compound 1：Different phytolaccanine A
1 compound 1 of table1H NMR and13C NMR data (DMSO-d6)
Compound 2：Phytolaccanine B
2 compound 2 of table1H NMR and13C NMR data (DMSO-d6)
Compound 3：(±) 3,3 '-dinor- rosin spirit
3 compound 3 of table1H NMR and13C NMR data (DMSO-d6)
Compound 4：7S,8R-americanin D
4 compound 4 of table1H NMR and13C NMR data (DMSO-d6)
Embodiment 2
(1) tung oil tree kind shell 1kg is dried, with 90% ethanol heating and refluxing extraction 3 times, the volume (L) of medicinal material weight (kg) and Extraction solvent is than for 1:5, recovered under reduced pressure extract obtains crude extract；
(2) extract Jing nonpolar macroporous adsorption resins obtained by step (1) are processed, with methyl alcohol and aqueous solvent：1:9,2:8,3:7,5:5,7:3 carry out gradient elution, receive 2:8,3:7,5:5,7:The eluent of 3 four flow points, reduced pressure concentration obtain crude oil Tong Zhong shell lignanoid；
(3) crude oil Tong Zhong shell lignanoid Jing polyamide column chromatographies obtained by step (2) are processed, with methyl alcohol and water mixed solvent：1:9,2:8,3:7,5:5,7:3 carry out gradient elution, collect and combine 2:8,3:7,5:5,7:3 four flow point eluents, reduced pressure concentration are dried, obtain refined tung oil tree Zhong Ke lignanoids.
The refined tung oil tree Zhong Ke lignanoids of gained, with embodiment 1, gained compound as reference substance, know, and confirms that its main lignanoid's spot is by the inspection of Jing thin-layer chromatographys：Different phytolaccanine A, phytolaccanine B, (±) 3,3 '-dinor- rosin spirit, 7S, 8R-americanin D, wherein different phytolaccanine A contents are 63.7%, phytolaccanine B content is 6.0%, (±) 3, and 3 '-dinor- rosin alcohol content is 14.9%, 7S, 8R-americanin D contents are 7.4%.
Embodiment 3
(1) tung oil tree kind shell 1.5kg is dried, with 70% ethanol heating and refluxing extraction 1 time, the volume (L) of medicinal material weight (kg) and Extraction solvent is than for 1:15, recovered under reduced pressure extract obtains crude extract；
(2) by extract Jing petroleum ethers obtained by step (1), ethyl acetate, extracting n-butyl alcohol 3 times, extractant is 1 with the volume ratio of extract:1, reduced pressure concentration ethyl acetate, butanol extraction liquid obtain crude oil paulownia kind shell total lignan；
(3) crude oil paulownia kind shell total lignan Jing silica gel column chromatographies obtained by step (2) are processed, with dichloromethane and methanol mixed solvent：100:1,100:5,100:20,100:50 carry out gradient elution, collect and combine containing 100:5,100:20 two flow point eluents, reduced pressure concentration are dried, obtain refined tung oil tree kind shell total lignan.
(4) tung oil tree kind shell total lignan is refined obtained by step (3), gained compound as reference substance, know, and confirms that its main lignanoid's spot is by the inspection of Jing thin-layer chromatographys with embodiment 1：Different phytolaccanine A, phytolaccanine B, (±) 3,3 '-dinor- rosin spirit, 7S, 8R-americanin D, wherein different phytolaccanine A contents are 70.9%, phytolaccanine B content is 5.5%, (±) 3, and 3 '-dinor- rosin alcohol content is 12.1%, 7S, 8R-americanin D contents are 6.0%.
Embodiment 4
(1) tung oil tree kind shell 3kg is dried, with 90% methyl alcohol heating and refluxing extraction 3 times, the volume (L) of medicinal material weight (kg) and Extraction solvent is than for 1:8, recovered under reduced pressure extract obtains crude extract；
(2) extract Jing hexamethylenes, chloroform, ethyl acetate obtained by step (1) are extracted 1 time, extractant is 3 with the volume ratio of extract:1, reduced pressure concentration chloroform and acetic acid ethyl acetate extract obtain crude oil paulownia kind shell total lignan；
(3) crude oil paulownia kind shell total lignan Jing silica gel column chromatographies obtained by step (2) are processed, with dichloromethane and methanol mixed solvent：100:1,100:4,100:10,100:50 carry out gradient elution, collect and combine 100:4,100:10 two flow point eluents, reduced pressure concentration are dried, obtain refined tung oil tree kind shell total lignan.
The refined tung oil tree kind shell total lignan of gained, with embodiment 1, gained compound as reference substance, know, and confirms that its main lignanoid's spot is by the inspection of Jing thin-layer chromatographys：Different phytolaccanine A, phytolaccanine B, (±) 3,3 '-dinor- rosin spirit, 7S, 8R-americanin D, wherein different phytolaccanine A contents are 62.5%, phytolaccanine B content is 6.4%, (±) 3,3 '-dinor- rosin alcohol content is 16.5%, 7S, and 8R-americanin D contents are 6.8%.
Embodiment 5
(1) tung oil tree kind shell 4kg is dried, with 70% methyl alcohol heating and refluxing extraction 2 times, the volume (L) of medicinal material weight (kg) and Extraction solvent is than for 1:10, recovered under reduced pressure extract obtains crude extract；
(2) by extract Jing petroleum ethers obtained by (1), ethyl acetate and extracting n-butyl alcohol 2 times, extractant is 2 with the volume ratio of extract:1, reduced pressure concentration ethyl acetate and butanol extraction liquid obtain crude oil paulownia kind shell total lignan；
(3) crude oil paulownia kind shell total lignan Jing polyamide column chromatographies mobile phase obtained by step (2) is second alcohol and water, and mixed solvent volume ratio is 1:9,3:7,6:4,8:2, ethanol/water volume ratio is 3:7,6:The tung oil tree kind shell total lignan that 4 eluted fractions must be refined.
Gained tung oil tree kind shell total lignan, with embodiment 1, gained compound as reference substance, know, and liquid phase analysis confirm that its main lignanoid's spot is by the inspection of Jing thin-layer chromatographys：Different phytolaccanine A, phytolaccanine B, (±) 3,3 '-dinor- rosin spirit, 7S, 8R-americanin D, wherein different phytolaccanine A contents are 66.4%, phytolaccanine B content is 5.3%, (±) 3, and 3 '-dinor- rosin alcohol content is 15.2%, 7S, 8R-americanin D contents are 6.5%.
Embodiment 6
The anti tumor activity in vitro test of prepared lignanoid in embodiment 3
(1), experiment material
Test medicine：3 gained tung oil tree kind shell total lignan of embodiment
Calf serum (is purchased from Hyclone)；Hyclone (TBD companies)；Tetramethyl azo azoles salt (MTT) U.S. Sigma (St.Louis, MO)；96 porocyte culture plates (Costar companies)；9 kinds of human lung carcinoma cells (A549, H226, H292, H460, H1792, SW1573, CALU-1, H1299, H358), a kind of SMMC-7721 HepG2, a kind of gastric carcinoma cell lines HGC-27 and a kind of chronic myeloid leukemia cell line K562 are purchased from Shanghai Kun Ken biochemical industries Co., Ltd.
(2), experimental technique
1. cell culture
The SMMC-7721 HepG2 in exponential phase, gastric carcinoma cell lines HGC-27 and chronic myeloid leukemia cell line K562 are taken, the dilution of above-mentioned cell is become 5 × 10 with the RPMI-1640 nutrient solutions containing 10% (v/v) hyclone3The cell suspending liquid of individual/L.Afterwards, it is inoculated in 96 orifice plates (100 μ L/ holes) so as to be placed in 37 DEG C, 5%CO248h is cultivated in environment.
2. medicine is prepared
Testing sample is dissolved with DMSO, is made into the mother liquor that concentration is 100mmol/L, is stored in -20 DEG C.Face the used time is diluted to 100 μ g/L, 50 μ g/L and 25 μ g/L and is tested with corresponding nutrient solution.When the sample of DMSO configurations is tested, final concentration of the 1 ‰ of DMSO.Positive drug cis-platinum (CDDP) and paclitaxel concentration are 50 μ g/L, 25 μ g/L and 1 μm of ol/L.Above-mentioned pastille nutrient solution is added in 96 orifice plates, is positioned in the incubator with cell culture the same terms and is cultivated 48h.
(3), experimental implementation process
Mtt assay operating process is as follows：
Mtt assay：
Data analysis is calculated in accordance with the following methods：
Inhibitory rate of cell growth=[1- administration groups OD value/control group OD values] × 100%.
Medicine half-inhibition concentration (IC is calculated using LOGIT methods50)。
(4) experimental result：It is shown in Table 5
The inhibitory activity that tung oil tree kind shell total lignan is grown to several human cancer cells in 5 embodiment 3 of table
CDDP (cis-platinum) and taxol are positive control medicine
As a result in showing embodiment 3, preparation-obtained tung oil tree kind shell has significant anti tumor activity in vitro, can significantly inhibit SMMC-7721 HepG2, and the growth of gastric carcinoma cell lines HGC-27 and chronic myeloid leukemia cell line K562 shows anti tumor activity in vitro.
Embodiment 7
The internal antitumor activity test of the tung oil tree kind shell total lignan prepared in embodiment 3
(1) experiment material
By test product：Tung oil tree kind shell total lignan
Cell line：Cell line S180 sarcoma (is provided by medicament teaching and research room of Shenyang Pharmaceutical University)
Animal subject：SPF level Kunming mouses (Kun Ming mice, KM mice), male, weight 18-22g are purchased from long-living Bioisystech Co., Ltd, quality certification number by Shenyang Pharmaceutical University's Experimental Animal Center：SCXK2010-0001.Animal freely ingests and drinks water, 23 ± 2 DEG C of environment temperature, humidity 30%-70%, lighting hours 12h/d (7:00～19:00 turns on light).Tested after raising 3 days.
Experiment reagent and instrument：
Endoxan (Hengrui Medicine Co., Ltd., Jiangsu Prov., lot number：14010925)
ACCULAB assay balances (Beijing Sai Duolisi instrument systems Co., Ltd)
T1000 electronic balances (Beijing Sai Duolisi instrument systems Co., Ltd)
(2), experimental technique
1. the foundation of knurl mouse model
S180 sarcoma knurl strain, inoculation is passed in abdominal cavity.When ascites well-grown, extract ascites cells out and count, adjustment cell concentration is 1 × 107Individual cell/mL, in right side of mice armpit hypodermic injection S180 sarcoma cell suspensions, per being only inoculated with 0.2ml.
2. treat and be grouped
40 mouse were randomly divided into 4 groups in the same day of inoculation.Respectively：Model control group, positive drug group, tung oil tree low dose group, tung oil tree high dose group.24h after inoculation, blank group gavage gives CMC-Na 10mL/kg, positive drug group, endoxan lumbar injection 25mg/kg, gavage gives 50mg/kg, 100mg/kg tung oil tree kind shell total lignan, successive administration 10 days respectively for tung oil tree low dosage, high dose group, put to death within 11st day, tumor mass, spleen, thymus gland are stripped, is weighed, calculate tumour inhibiting rate and organ index respectively.
Tumour inhibiting rate (%)=(the average knurl weight of the average knurl weight-administration group of model group) average knurl weight × 100% of/model group
Organ index (%)=organ weights/Mouse Weight × 100%
3. statistical method
Data are analyzed using SPSS17.0 statistical softwares.As a result represented using mean value ± standard error (Mean ± S.E.), globality difference, P are evaluated using One-Way ANOVA (One-Way Analysis of variance, one-way analysis of variance)<0.05 is have significant difference.
(3), experimental result
As a result show, compared with model group, tung oil tree kind shell total lignan high dose group significantly reduces knurl weight (P<0.05), and tumour inhibiting rate be 54.7%；And the average knurl weight of endoxan group significantly reduces (P<0.01), tumour inhibiting rate is 66.8%.In terms of organ index：Compared with model group, endoxan group significantly reduces mouse spleen, thymus index (P<0.05,P<0.01,P<0.001)；And the organ index of tung oil tree kind shell total lignan low dose group and high dose there was no significant difference.
Impact (Mean ± S.D., n=8) of the 6 tung oil tree kind shell total lignan of table to mouse S180 tumours and organ index
Note：*P<0.05,**P<0.01,***P<0.001 compared with model group；
Embodiment 8
The anti-neuroinflamation active testing of the tung oil tree kind shell total lignan prepared in embodiment 3
(1), experiment material
By test product：Tung oil tree kind shell crude extract and refined tung oil tree kind shell total lignan.
Experimental cell strain and source：BV2：Mouse microglia strain.
Experiment reagent and instrument：
7 experiment reagent of table and instrument
| IMDM culture mediums | Gibco |
| Hyclone | Gibco BRL lot numbers 1339728 |
| LPS(E5:055) | U.S.'s Sigma lot numbers：069k4005 |
| Tetramethyl azo azoles salt (MTT) | U.S.'s Sigma lot numbers：111108 |
| Dimethyl sulfoxide (DMSO) (DMSO) | Shenyang chemical reagent factory |
| ELIASA | Austrian TECAN |
| 96 porocyte culture plates | Costar companies |
(2), experimental technique
1. the culture of mouse microglia system BV2
All glasswares and metallic weapon (blake bottle, pipette, solution bottle etc.) used in cell culture and model foundation, through 121 DEG C of autoclaving 30min, thoroughly to remove the LPS of depollution.The cell culture fluid for including 5% hyclone and 50 μ g/mL 2 mercapto ethanols is configured to based on IMDM culture mediums.Microglia is with about 4 × 105The concentration of cells/mL is in 5%CO2, Secondary Culture in 37 DEG C of blake bottles accounts for blake bottle floor space 50-60% to the 3rd day attached cell, digests attached cell with pancreatin, is passaged to another blake bottle.Using the BV2 after -80 DEG C of ultra low temperature freezer cryopreservation resuscitations as the first generation, 3-8 is selected to be tested for BV2 cells.
2. method for preparation of drug
By tung oil tree kind shell crude extract and total lignan, dissolved with DMSO.Mother liquor (100 μM or 100mg/mL) is made into, -20 DEG C are stored in.Facing the used time is diluted with IMDM nutrient solutions, and crude extract is diluted to 100,30,10,3,1 μ g/mL, DMSO final concentrations successively<1‰.
3. Griess methods detection compound activates the inhibitory action of microglia to LPS[1 , 3]
Take the logarithm the BV2 microglias in growth period, cell density is adjusted to into 5 × 10 with the fresh IMDM culture mediums containing 5% hyclone5Cells/mL, is inoculated in 96 orifice plates, 100 μ L/well, in 37 DEG C, 5%CO2Incubator in culture.Change the fresh medium of serum-free after cell attachment culture 24h into, while carrying out agent-feeding treatment.Tung oil tree kind shell crude extract and total lignan set 100,30,10,3,1 μ g/mL and LPS collective effects of dosage.Set blank simultaneously.The final concentration of 100ng/ml of LPS in each administration group.After continuing culture 24h after cell dosing, supernatant, NO in Griess colorimetric determination supernatants are collected2-Content.
4. impact of the mtt assay detection compound to microglia survival rate[2]
Take the logarithm the BV2 microglias of growth period culture, cell density is adjusted to into 5 × 10 with containing the fresh IMDM culture mediums of 5% hyclone5Cells/mL, is inoculated in 96 orifice plates, 100 μ L/well, in 37 DEG C, 5%CO2Incubator in culture.Fresh medium is changed into after cell attachment culture 24h, while carrying out agent-feeding treatment.Tung oil tree kind shell total lignan sets 100,30,10,3,1 μ g/mL and LPS collective effects of dosage.The final concentration of 100ng/mL of LPS in each administration group.Continuing culture 24h after cell dosing, then MTT solution being added in cell liquid, 10 μ L/well, the common incubation 3h at 37 DEG C by cell and 0.25mg/mL MTT absorb nutrient solution, be subsequently adding the DMSO solution of 150 μ L, determine its optical density OD value.Data processing, carries out data processing using ELIASA corresponding software, calculates the mean value of 3 hole OD values of each sample, is calculated as follows cell survival rate (cell viability, CV%) using mean value.
Mean value × 100% of the mean value/blank control group OD value of cell survival rate %=sample sets OD values
5. statistical method
Whole data are tested analysis using SPSS (13.0) statistical package.As a result being represented with mean value ± standard error, evaluate globality difference, mean carries out homogeneity of variance analysis using One-Way ANOVA analytic approach between group, and combine Dunnett ' s test analysis methods carries out comparing between group.Multisample homogeneity test of variance is checked using Levene, works as p>0.05, variance is difference that is neat, checking multigroup mean using Dunnett ' s bilateral T, works as p<0.05, heterogeneity of variance checks the difference of multigroup mean using Dunnett T3.
⑥IC50Computational methods[4]
The parameters such as each dosage and inhibiting rate are calculated into IC with nonlinear regression and fitting50。
(3), experimental result and discussion
8 experimental result of table
| Sample | IC50 | MINO |
| Tung oil tree kind shell crude extract | 17.22 | 48.82 |
| Tung oil tree kind shell refines lignanoid | 10.7 | 48.82 |
As a result show, tung oil tree kind shell total lignan has the effect for suppressing the BV2 cells of LPS inductions to discharge NO well.
Above-described embodiment technology design only to illustrate the invention and feature, its object is to allow person skilled in the art will appreciate that present disclosure and implement according to this, can not be limited the scope of the invention with this.All equivalence changes made according to spirit of the invention or modification, should all be included within the scope of the present invention.
Claims (10)
1. the lignans extract for extracting in a kind of kind shell from tung oil tree, it is characterised in that main by different business
Lu Su A, phytolaccanine B, (±) 3,3 '-dinor- rosin spirit, 7S, 8R-americanin D compositions.
2. lignans extract according to claim 1, it is characterised in that：The lignanoid extracts
In thing, the percentage by weight of each compound is：Different phytolaccanine A >=62%, phytolaccanine B >=5%, (±) 3,
3 '-dinor- rosin spirit >=12%, 7S, 8R-americanin D >=6%.
3. the preparation method of lignans extract described in a kind of claim 1, it is characterised in that include
Following steps：
(1) tung oil tree kind shell, is dried, after crushing, uses solvent extraction, recovered under reduced pressure extract to obtain crude extract；
(2), by the process of said extracted thing Jing nonpolar macroporous adsorption resins, gradient is carried out with solvent and washed
It is de-, the eluent containing lignanoid is collected and combined, reduced pressure concentration obtains tung oil tree kind shell total lignan；
Or said extracted liquid is used successively petroleum ether or hexamethylene, dichloromethane or chloroform, ethyl acetate,
Extracting n-butyl alcohol, reduced pressure concentration extract obtain total lignan；
(3), the total lignan Jing polyamide column chromatographies obtained by step (2) are processed, and carry out ladder with solvent
Degree wash-out, collects and combines the eluent containing lignanoid, and reduced pressure concentration is dried, obtains refined lignanoid；
Or by the process of tung oil tree total lignan Jing silica gel column chromatographies, gradient elution is carried out with organic solvent, receive
Collection merges the eluent of oil-containing paulownia lignanoid, reduced pressure concentration, dry must refine tung oil tree lignanoid.
4. the preparation method of lignans extract according to claim 3, it is characterised in that：Step
(1) in, Extraction solvent is ethanol that volumetric concentration is 60%～100% or methyl alcohol, extraction time is 1～
3 times, the volume ratio of medicinal material weight and Extraction solvent is 1:5～1:15, wherein unit of weight is kg, body
Product unit is L.
5. the preparation method of lignans extract according to claim 3, it is characterised in that：Step
(2) in, the mobile phase of macroporous adsorbent resin chromatography is that methyl alcohol and water mixed solvent or the mixing of second alcohol and water are molten
Agent, in mixed solvent, the volume ratio of methyl alcohol or second alcohol and water is 1：9～8：2；
The number of times that extract is extracted successively in step (2) is 1-3 time, the body of extractant and extract
Product is than being 1:1～3:1.
6. the preparation method of lignans extract according to claim 3, it is characterised in that：Step
(3) in, polyamide column chromatography mobile phase is methyl alcohol and water mixed solvent or ethanol and water mixed solvent, is mixed
In bonding solvent, the volume ratio of methyl alcohol or second alcohol and water is 1：9～8：2.
7. the preparation method of lignans extract according to claim 3, it is characterised in that：Step
(3) in, silica gel column chromatography mobile phase is dichloromethane and methyl alcohol, chloroform and methanol mixed solvent, is mixed
In solvent, the volume ratio of dichloromethane or chloroform and methyl alcohol is 100：1～2：1.
8. application of the lignans extract described in claim 1 in terms for the treatment of neuroinflammatory conditions medicine.
9. application of the lignans extract described in claim 1 in terms of cancer therapy drug is prepared.
10. lignans extract described in claim 1 is being prepared with cancer cell specific induction of apoptosis effect
Application in terms of medicine.
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| CN109369665A (en) * | 2018-11-30 | 2019-02-22 | 中南林业科技大学 | The method of double tetrahydrofuran lignans is extracted from the tea of South Mountain |
| CN110964025A (en) * | 2018-09-29 | 2020-04-07 | 沈阳药科大学 | Lignan compound and preparation method and application thereof |
| CN112067726A (en) * | 2020-09-24 | 2020-12-11 | 黑龙江珍宝岛药业股份有限公司 | Method for determining total lignans in Shuxuening injection |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110964025A (en) * | 2018-09-29 | 2020-04-07 | 沈阳药科大学 | Lignan compound and preparation method and application thereof |
| CN110964025B (en) * | 2018-09-29 | 2021-07-30 | 沈阳药科大学 | Lignan compound and preparation method and application thereof |
| CN109369665A (en) * | 2018-11-30 | 2019-02-22 | 中南林业科技大学 | The method of double tetrahydrofuran lignans is extracted from the tea of South Mountain |
| CN112067726A (en) * | 2020-09-24 | 2020-12-11 | 黑龙江珍宝岛药业股份有限公司 | Method for determining total lignans in Shuxuening injection |
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