CN105294616A - New limonoids for protecting endothelial cells - Google Patents
New limonoids for protecting endothelial cells Download PDFInfo
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Abstract
The invention discloses new limonoids for protecting endothelial cells. The compound is reported for a first time, and limonoids have a novel structure; the compound can be obtained from dried and matured toosendan fruits by extraction, separation and purification. External tests prove that the compound can obviously improve viability of human umbilical vein endothelial cells which are damaged by ox-LDL, substantially reduce HUVEC apoptosis induced by ox-LDL, and the effect appears dose-dependent. External tests prompt the compound (I) can realize the endothelial protection effect by inhibiting the HUVEX apoptosis induced by ox-LDL, and can be further used for developing a medicament for protecting endothelial cells.
Description
Technical field
The invention belongs to technical field of pharmaceuticals, be specifically related to from the dry mature fruit of Szechwan Chinaberry Fruit, be separated obtain a kind of and there is limonoid of endotheliocyte provide protection and preparation method thereof.
Background technology
Szechwan Chinaberry Fruit (having another name called Fructus Toosendan) is the dry mature fruit of Meliaceae plant melia toosendan MeliatoosendanSieb.etZucc., begin to be loaded in Shennong's Herbal, gather during fruit maturation in winter, for tradition regulate the flow of vital energy, insect repellent, be distributed widely in the ground such as Sichuan, Hunan, Hubei, Guizhou, superior with the product person in Sichuan, therefore named is Szechwan Chinaberry Fruit.Its mildly bitter flavor, slightly poisonous, have soothing the liverly to expel the heat-evil, the effect such as promoting the circulation of QI to relieve pain, desinsection, be applicable to the diseases such as pathogenic fire derived from stagnation of liver-QI, the chest side of body, abdominal distention, hernia pain, abdominal pain due to worm stagnation, have clinically and apply comparatively widely.
According to research reports, containing number of chemical composition in Szechwan Chinaberry Fruit, the compounds such as terpene, volatile oil, obacalactone, flavones, lipid acid, phenolic acids and polysaccharide are mainly comprised.Wherein triterpenes components Toosendanin is the characteristic chemical constituent of melia plant, is again the toxic component of Szechwan Chinaberry Fruit and the effective constituent of expelling parasite.Szechwan Chinaberry Fruit volatilization main body of oil comprises unsaturated organic acid, alcohols, aldoketones, ester class, and the volatile oil component of its processed product obviously reduces relative to raw product, and the relative content of unsaturated organic acid obviously increases.
Szechwan Chinaberry Fruit is very extensive as traditional Chinese medicine clinical application, and the domestic and international research to its pharmacological action at present mainly concentrates on chinaberry alkane type triterpenoid and limonoid.Research show Szechwan Chinaberry Fruit have expelling and killing ascarid, antitumor, antiviral, respiration inhibition, anti-oxidant, suppress the effect such as osteoclast, analgesia.
Summary of the invention
The object of this invention is to provide and a kind ofly from the dry mature fruit of Szechwan Chinaberry Fruit, be separated obtain a kind of there is limonoid of endotheliocyte provide protection and preparation method thereof.
Above-mentioned purpose of the present invention is achieved by technical scheme below:
There is the compound (I) of following structural formula,
The preparation method of described compound (I), comprise following operation steps: the dry mature fruit of Szechwan Chinaberry Fruit is pulverized by (a), extract with 70 ~ 80% alcohol heat reflux, united extraction liquid, be concentrated into without alcohol taste, use sherwood oil, ethyl acetate and water saturated n-butanol extraction successively, obtain petroleum ether extract, acetic acid ethyl ester extract and n-butyl alcohol extract respectively; Acetic acid ethyl ester extract macroporous resin removal of impurities in (b) step (a), first use 10% ethanol elution, 8 column volumes, then use 80% ethanol elution, 10 column volumes, collect 80% ethanol eluate, concentrating under reduced pressure obtains 80% ethanol elution thing medicinal extract; C in () step (b), 80% ethanol elution medicinal extract purification on normal-phase silica gel is separated, obtain 5 components successively with the methylene chloride-methanol gradient elution that volume ratio is 75:1,45:1,25:1,15:1 and 1:1; D in () step (c), component 4 is separated further by purification on normal-phase silica gel, obtain 3 components successively with the methylene chloride-methanol gradient elution that volume ratio is 20:1,15:1 and 8:1; E in () step (d), component 2 reverse phase silica gel of octadecylsilane bonding is separated, with the methanol aqueous solution isocratic elution that concentration expressed in percentage by volume is 70%, collect 10 ~ 12 column volume elutriants, elutriant concentrating under reduced pressure obtains pure compound (I).
Further, described macroporous resin is D101 macroporous adsorbent resin.
Further, described alcohol heat reflux extracts the alcohol concn adopted is 75%.
A kind of pharmaceutical composition, the described compound (I) wherein containing treatment significant quantity and pharmaceutically acceptable carrier.
The application of described compound (I) in the medicine preparing endotheliocyte protection.
The application of described pharmaceutical composition in the medicine preparing endotheliocyte protection.
When the compounds of this invention is used as medicine, directly can uses, or use with the form of pharmaceutical composition.
This pharmaceutical composition contains the compounds of this invention (I) for the treatment of significant quantity, and all the other are acceptable on pharmacology, nontoxic to humans and animals and pharmaceutically acceptable carrier of inertia and/or vehicle.
Described pharmaceutically acceptable carrier or vehicle are that one or more are selected from solid, semisolid and liquid diluent, filler and pharmaceutical preparation assistant agent.Pharmaceutical composition of the present invention is used with the form of per weight dose.Medicine of the present invention is applied to by form that is oral or injection the patient needing treatment.For time oral, tablet, slow releasing tablet, controlled release tablet, capsule, dripping pill, micropill, suspensoid, emulsion, powder or granule, oral liquid etc. can be made into; During for injecting, can be made into water-based or oily solution, aseptic powder injection, liposome or the emulsion etc. of sterilizing.
Accompanying drawing explanation
Fig. 1 is compound (I) structural formula;
Fig. 2 is that the theoretical ECD value of compound (I) compares with experiment ECD value.
Embodiment
Further illustrate essentiality content of the present invention below in conjunction with embodiment, but do not limit scope with this.Although be explained in detail the present invention with reference to preferred embodiment, those of ordinary skill in the art should be appreciated that and can modify to technical scheme of the present invention or equivalent replacement, and does not depart from essence and the scope of technical solution of the present invention.
Embodiment 1: compound (I) is separated preparation and structural identification
Reagent source: ethanol, sherwood oil, ethyl acetate, propyl carbinol, methylene dichloride are analytical pure, purchased from Shanghai Ling Feng chemical reagent company limited, methyl alcohol, analytical pure, purchased from Jiangsu Han Bang chemical reagent company limited.
Preparation method: the dry mature fruit (10kg) of Szechwan Chinaberry Fruit is pulverized by (a), (25L × 3 time) are extracted with 75% alcohol heat reflux, united extraction liquid, be concentrated into without alcohol taste (3L), use sherwood oil (3L × 3 time), ethyl acetate (3L × 3 time) and water saturated propyl carbinol (3L × 3 time) to extract successively, obtain petroleum ether extract, acetic acid ethyl ester extract (431g) and n-butyl alcohol extract respectively; Acetic acid ethyl ester extract D101 macroporous resin removal of impurities in (b) step (a), first use 10% ethanol elution, 8 column volumes, use 80% ethanol elution, 10 column volumes again, collect 80% ethanol eluate, concentrating under reduced pressure obtains 80% ethanol elution thing medicinal extract (161g); C in () step (b), 80% ethanol elution medicinal extract purification on normal-phase silica gel is separated, successively with volume ratio be 75:1 (8 column volumes), the methylene chloride-methanol gradient elution of 45:1 (8 column volumes), 25:1 (8 column volumes), 15:1 (10 column volumes) and 1:1 (5 column volumes) obtains 5 components; D component 4 (52g) is separated further by purification on normal-phase silica gel in () step (c), successively with volume ratio be 20:1 (8 column volumes), the methylene chloride-methanol gradient elution of 15:1 (10 column volumes) and 8:1 (8 column volumes) obtains 3 components; E in () step (d), component 2 (26g) reverse phase silica gel of octadecylsilane bonding is separated, with the methanol aqueous solution isocratic elution that concentration expressed in percentage by volume is 70%, collect 10-12 column volume elutriant, elutriant concentrating under reduced pressure obtains pure compound (I) (37mg).
Structural identification: HR-ESIMS shows [M+Na]
+for m/z683.3010, can obtain molecular formula in conjunction with nuclear-magnetism feature is C
35h
48o
12, degree of unsaturation is 12.Hydrogen nuclear magnetic resonance modal data δ
h(ppm, DMSO-d
6, 600MHz): H-1 (5.42, d, J=4.0), H-2 (2.39, dd, J=15.0, 4.0), H-2 (2.86, d, J=15.0), H-5 (2.33, d, J=7.5), H-6 (2.14, m), H-6 (3.15, d, J=18.9), H-9 (3.32, d, J=7.6), H-11 (5.28, dd, J=11.0, 7.6), H-12 (5.62, d, J=11.0), H-15 (3.66, s), H-16 (1.69, m), H-16 (2.09, m), H-17 (2.88, dd, J=10.5, 7.3), H-18 (0.83, s), H-19 (1.55, s), H-21 (6.97, s), H-22 (6.04, s), H-23 (7.16, s), H-28 (0.91, s), H-29 (1.02, s), H-30 (4.98, s), H-30 (5.27, s), 7-OCH
3(3.47, s), 1-OAc (1.86, s), 11-OAc (1.90, s), H-2 ' (1.98, m), H-3 ' (0.80, d, J=7.0), H-4 ' (0.81, d, J=7.0), carbon-13 nmr spectra data δ
c(ppm, DMSO-d
6, 150Hz): 77.7 (CH, 1-C), 36.8 (CH
2, 2-C), 211.6 (C, 3-C), 49.0 (C, 4-C), 44.1 (CH, 5-C), 31.3 (CH
2, 6-C), 174.5 (C, 7-C), 134.1 (C, 8-C), 50.2 (CH, 9-C), 46.1 (C, 10-C), 71.7 (CH, 11-C), 74.1 (CH, 12-C), 45.2 (C, 13-C), 70.7 (C, 14-C), 59.1 (CH, 15-C), 33.3 (CH
2, 16-C), 37.2 (CH, 17-C), 12.9 (CH
3, 18-C), 16.7 (CH
3, 19-C), 122.0 (C, 20-C), 139.6 (CH, 21-C), 110.7 (CH, 22-C), 142.2 (CH, 23-C), 24.6 (CH
3, 28-C), 24.1 (CH
3, 29-C), 123.0 (CH
2, 30-C), 51.4 (CH
3, 7-OCH
3), 169.6 (C, 1-OAc), 19.8 (CH
3, 1-OAc), 174.5 (C, 11-OAc), 21.2 (CH
3, 11-OAc), 175.5 (C, 1 '-C), 33.7 (CH, 2 '-C), 18.0 (CH
3, 3 '-C), 18.1 (CH
3, 4 '-C), carbon atom mark is see Fig. 1.Infrared spectra shows that this compound contains hydroxyl (3441cm
-1) and carbonyl (1735cm
-1) group.
13cNMR spectrum and DEPT spectrum show 35 carbon signals, comprising the methyl of nine, four methylene radical (an alkene carbon), 11 methyne (three alkene carbon, four containing oxygen carbon), and 11 quaternary carbons (containing oxygen carbon for two alkene carbon, five carbonyl carbon).In addition, this compound contains the monosubstituted furan nucleus of a β [δ C122.0 (C-20), 139.6 (C-21), 110.7 (C-22) and 142.2 (C-23)], according to H-17 (δ H2.88, dd, J=10.5 in HMBC spectrum, 7.3) with the dependency of C-21 and C-22, deducibility furan nucleus is positioned on C-17 position.The limonoid that this compound known is the open loop of a B ring is analyzed by nuclear magnetic data.In addition, this compound contains an isobutyl acyl-oxygen based structures.Me-3 ' (0.80, d, J=7.0Hz), Me-4 ' (0.81, d, J=7.0Hz) and H-2 ' in HMBC spectrum (1.98, m) and the above-mentioned inference of the relevance verification of C-1 ' (δ C175.5).Me-18 and C-12 in being composed by HMBC; The intersection peak known isobutyl acyl-oxygen substrate section of H-12 and C-1 ' is positioned on C-12 position.In HMBC spectrum, Me-19 (δ H1.55, s) and H-9 (δ H3.32, d, J=7.6Hz) and C-1 (δ C77.7); The dependency of H-1 (δ H5.42, d, J=4.0Hz) and H-11 (δ H5.28, dd, J=11.0,7.6Hz) its corresponding acetyl carbonyl (δ C169.6,174.5), and
1h-
1h-1 and H in HCOSY spectrum
2-2 (δ H2.39, dd, J=15.0,4.0Hz; 2.86, d, J=15.0) intersection peak, show that C-1 and C-11 (δ C71.7) position is connected with an acetoxyl group respectively.In HMBC spectrum, with the dependency of C-3 (δ C211.6), H-1, Me-28 (δ H0.91, s) and Me-29 (δ H1.02, s) show that C-3 is ketone carbonyl.In ROESY spectrum, Me-19 and Me-29 and H-12; And H-12 and H-17 dependency shows that they are beta comfiguration.In addition, H-9 (δ H3.32, d, J=7.6Hz) and H-1 in ROESY spectrum; H-9 and H-11; H-9 and H-5 (δ H2.33, d, J=7.5Hz); The intersection peak of H-5 and Me-28 and H-11 and Me-18 (δ H0.83, s) shows that they are α-configuration.These results and key proton (J
9,11=7.6Hz and J
11,12=11.0Hz) between larger coupling constant match.Comprehensive hydrogen spectrum, carbon spectrum, HMBC spectrum and ROESY spectrum, and document is about correlation type nuclear magnetic data, can substantially determine this compound as shown in Figure 1, steric configuration is determined further by ECD test, theoretical value and experimental value basically identical (Fig. 2).
Embodiment 2: compound (I) pharmacological action is tested
One, material and instrument
Human umbilical vein endothelial cells pearl (HUVEC) is provided by Shanghai Wu Li Bioisystech Co., Ltd cell bank.Compound (I) is made by oneself, and HPLC normalization method purity is greater than 98%.RPMI-1640 substratum, MTT, dimethyl sulfoxide (DMSO) are all purchased from Sigma company.Foetal calf serum is purchased from HyCLone company.FITC marks goat anti-rabbit igg, the anti-human factor Ⅷ related antigen of rabbit is purchased from Beijing Bo Aosen Bioisystech Co., Ltd.MDA reagent box for detecting content is purchased from Nanjing and builds up biological study institute.AnnexinV-FITC apoptosis detection kit is purchased from Centrebio company.Ethylenediamine tetraacetic acid (EDTA) (EDTA) is purchased from Guangzhou Wei Jia Science and Technology Ltd..
DK-8AD type electric heating constant temperature tank (Shanghai Yiheng Scientific Instruments Co., Ltd), ER-120A type electronic analytical balance (Shimadzu Corporation), 6219 type electronics pH meters (Shanghai Ren Shi Electronics Co., Ltd.), high speed low temperature centrifugal machine (Sigma company), L420 table-type low-speed self-poise whizzer (Xiang Yi whizzer Instrument Ltd.), SYC-2101 horizontal shaker (its woods Bel instrument manufacturing company limited), 1000 μ L micro sample-adding rifle × 1, 20-100 μ L micro sample-adding rifle × 1, 1-20 μ L micro sample-adding rifle × 1 (French Ji Ersen company), CO
2(incubator Heraeus company), super clean bench (safe and sound company of Su Jing group), inverted phase contrast microscope (German Lycra company), microplate reader (Sigma company), FACSCaLibur (flow cytometer).
Two, test method
1, the cultivation of huve cell
1.1 culture condition
The HUVEC cell strain newly bought is inoculated in the RPMI-1640 substratum containing 10% foetal calf serum.Be placed in 37 DEG C, 5%CO
2in incubator, within every 2 days, change 1 subculture.
1.2 passage
Getting one bottle of HUVEC observation of cell under inverted phase contrast microscope, as grown up to fine and close individual layer, can go down to posterity; Shaken gently by culturing bottle for several times, suspended and floated over the fragment of cell surface, then poured out together with nutrient solution, draw 2 ~ 3mLPBS liquid and add in culturing bottle, hypsokinesis of vibrating gently is gone, and repeats 3 times; Add 1mL0.25% pancreatin, be limited can cover at the bottom of bottle, rotate culturing bottle, make moistening whole cellular layer, put 37 DEG C of digested 2 ~ 3min of incubator, till oneself contraction of cell to be confirmed becomes bowlder; Add 1mL nutrient solution in culturing bottle, stop digestion, draw nutrient solution with suction pipe and repeatedly blow and beat bottle parietal cell gently, make it to depart from from bottle wall to form cell suspension, inject centrifuge tube centrifugal (800r/min, 5min) abandoning supernatant afterwards; Add 3mL nutrient solution in centrifuge tube, draw nutrient solution with suction pipe and blow and beat gently for several times, make cell suspending weight, then press 1:3 or 1:4 and distribute Secondary Culture; Be placed in 37 DEG C, 5%CO in incubator
2cultivate in thermostat container, after 24h, change liquid, within usual 3-4 days, can individual layer be formed, now just interchangeable maintenance medium for experiment.
The counting of 1.3HUVEC
First get cell suspension 50 μ L to be measured to instill in cell counting count board, by white blood cell count(WBC) method, under low power lens, count the total cellular score in 4 block plaid of 4 jiaos, then with following formulae discovery cell concn: viable count/4 × 10 in 4 block plaid
4=cell count/mL
2, the qualification of huve cell
Sterile cover slips 1cm × 1cm is placed, by endotheliocyte suspension inoculation on cover glass, when endotheliocyte grows to converging state in 6 well culture plates, take out cover glass, with PBS flush cover slide, put into 100% acetone and fix 15min, rinse 3 times with PBS, each 2min, drip the H of 3%
2o
2incubated at room 8min, PBS rinse 3 times, each 2min, drip rabbit anti-human boron factor monoclonal antibodies (1:100), another cover glass do not add primary antibodie and make negative control 4 DEG C and spend the night.Next day, rinse 3 times with PBS, each 2min, drip the goat anti-rabbit igg of FITC mark one note, hatch 60min for 37 DEG C, rinse 3 times with PBS, each 2min, fluorescence microscopy Microscopic observation is also taken pictures.
3, the cytoactive of MTT colorimetric method for determining HUVEC is utilized
3.1 utilize MTT colorimetry to observe different dense compound (I) to the impact of huve cell activity
3.1.1 Secondary Culture
By HUVEC by 2 × 10
4the density of/mL is with the RPMI-1640 culture medium inoculated containing 10%FBS in 96 orifice plates, and 200 μ L are planted in every hole.
3.1.2 to divide into groups dosing
After passage cell cultivates 24h, with the RPMI-1640 substratum compounding pharmaceutical containing 10%FBS, cell is divided into 6 groups at random: control group (control), compound (I) 5 μ g/mL group, compound (I) 10 μ g/mL group, compound (I) 20 μ g/mL group, compound (I) 40 μ g/mL group, compound (I) 80 μ g/mL group, hole, every hole 6.Continue cultivation after 48 hours, mtt assay detects cytoactive.
3.1.3MTT method detects
Add oneself MTT solution of preparing of 20 μ L directly to every hole, hatch 4h for 37 DEG C, exhaustion supernatant, then adds 150 μ LDMSO.After abundant dissolving to be crystallized, microplate reader is utilized to measure OD value at wavelength 490nm place.
3.2 utilize MTT colorimetry to observe different concns ox-LDL to the impact of huve cell activity
3.2.1 Secondary Culture: the same.
3.2.2 to divide into groups dosing
After cell cultures 24h, with the RPMI-1640 substratum compounding pharmaceutical containing 10%FBS, cell is divided into 6 groups at random: control group (control), ox-LDL (for self-control) 10 μ g/mL groups, ox-LDL20 μ g/mL group, ox-LDL40 μ g/mL group, ox-LDL80 μ g/mL group, ox-LDL160 μ g/mL group, hole, every hole 6.Continue cultivation 24 hours, mtt assay detects cytoactive.
The intervention effect that 3.3 utilize MTT colorimetry to observe compound (I) induces huve cell to damage to ox-LDL
3.3.1 Secondary Culture: the same.
3.3.2 to divide into groups dosing
After cell cultures 24h, with the RPMI-1640 substratum compounding pharmaceutical of 10%FBS, cell is divided into 6 groups at random: control group (control), ox-LDL50 μ g/mL group, ox-LDL50 μ g/mL+ compound (I) 5 μ g/mL group, ox-LDL50 μ g/mL+ compound (I) 10 μ g/mL group, hole, ox-LDL50 μ g/mL+ compound (I) 20 μ g/mL every hole 6.Continue cultivation after 24 hours, mtt assay detects cytoactive.
4, utilizing Flow Cytometry to observe compound (I) induces the intervention of huve cell apoptosis to do to ox-LDL
4.1 use Secondary Culture
By HUVEC by 2 × 10
5the density of/mL is with the RPMI-1640 culture medium inoculated containing 10%FBS in 6 orifice plates, and 1500 μ L are planted in every hole.
4.2 grouping dosings
After cell cultures 24h, with RPMI-1640 serum free medium serum deprivation 24h, cell is made to enter stationary state.With containing RPML-1640 blood serum medium compounding pharmaceutical, cell is divided into 5 groups at random: control group (control), ox-LDL50 μ g/mL group, ox-LDL50 μ g/mL+ compound (I) 5 μ g/mL group, ox-LDL50 μ g/mL+ compound (I) 10 μ g/mL group, the every hole of ox-LDL50 μ g/mL+ compound (I) 20 μ g/mL adds 1mL pastille substratum.
4.3 operation steps
After dosing, 12h draws materials, and cell culture fluid sucking-off in centrifuge tube, PBS washs attached cell once, with pancreatin cell dissociation buffer peptic cell.After cell dissociation gets off, transfer in centrifuge tube, PBS washes three times (centrifugal 10 minutes of 1000g).Add 200 μ L binding buffer liquid, re-suspended cell.Add 10 μ LAnnexinv-FITC, 10 μ LPI staining fluids mix gently.Room temperature lucifuge hatches 15 minutes, upper machine testing apoptosis in 30 minutes.
5, statistical analysis
Adopt SPSS13.0 to add up a software to analyze, result is expressed as: mean scholar standard deviation (
).When variance is neat, two groups are compared and adopt LSD, to compare more uses Dunnett to check as organized with control group, when heterogeneity of variance, and employing WeLch robust iterative, then adopt T3 method to compare between two.P<0.05 indicates statistical significance.
Three, result and conclusion
1, different concns compound (I) is on the impact of HUVEC cell viability
Compound (I) medicine is in certain its pharmacological action of concentration range competence exertion, and different cell systems also can be different to the susceptibility of medicine.For this reason, the drug level scope of experiment is first determined with mtt assay.Result is as table 1 (VS control group
#p<0.01) show, the effects of action of compound (I) Human Umbilical Vein Endothelial Cells of different concns is different, and drug level (5 ~ 20 μ g/mL) cytoactive of low dosage compares with control group, does not have significant difference.40 μ g/mL act on 48 hours later cell vigor and have dropped about 50%, and 80 μ g/mL act on 48 hours later cell vigor and have dropped 70%, compare and have significant difference (P<0.01), create certain toxic side effect with control group.Therefore this tests compound (I) concentration used is 5,10,20 μ g/mL, to get rid of the toxic action of medicine self to cell.
2, different concns ox-LDL is on the impact of huve cell vigor
Due to the difference of Ox LDL degree of oxidation, the cytotoxicity scope of Ox LDL can be different.Therefore we first adopt MTT to determine the cytotoxic scope of ox-LDL.Result is as table 2 (VS control group
*p<0.05
#p<0.01), shown in, different concns ox-LDL is different on the impact of HUVEC cytoactive.Compared with control group, 10 μ g/mL groups have the trend promoting that HUVEC cytoactive increases, but compare with control group and do not have significant difference (P>0.05); 20 ~ 100 μ g/mL respectively organize endothelial cell activity to be increased with concentration and reduces, and compares have significant difference (P<0.05 or P<0.01) with control group.
3, compound (I) induces the intervention effect of huve cell damage to ox-LDL
Result is as table 3 (VS.ox-LDLgroup
#p<0.01) shown in, the cytoactive that ox-LDL (50 μ g/mL) organizes declines, compare with control group and have significant difference (P<0.01), illustrate that ox-LDL group cytoactive significantly reduces, ox-LDL (50 μ g/mL) has damaging action to normal HUVEC.The cytoactive of compound (I) various dose group is all higher than ox-LDL group, statistical significance (P<0.01) is had with ox-LDL group comparing difference, illustrate that the cytoactive of these three dosage groups is significantly higher than ox-LDL group, the HUVEC damage that prompting compound (I) can suppress ox-LDL to induce.The cytoactive of compound (I) 10 μ g/mL and compound (I) 20 μ g/mL group, higher than compound (I) 5 μ g/mL group, compares with compound (I) 5 μ g/mL and has significant difference (P=0.01 or P<0.01).Compound (I) 20 μ g/mL cytoactive comparatively compound (I) 10 μ g/mL group has rising trend, but does not have significant difference (P=0.185).The Endothelium Protective effect of prompting compound (I) has dose-effect relationship within the specific limits.
4, compound (I) is on the impact of the huve cell apoptosis of ox-LDL induced damage
Each group of fluidic cell result display: the apoptosis rate difference of each group cell has statistical significance (P<0.01), the proliferation index of ox-LDL group obviously raises, compare with control group have significant difference (P<0.01) to illustrate ox-LDL (50 μ g/mL) has damaging action to normal HUVEC.The apoptosis rate of compound (I) various dose group is all lower than ox-LDL group, statistical significance (P<0.01) is had with ox-LDL group comparing difference, illustrate that the apoptosis rate of these three dosage groups is significantly lower than ox-LDL group, the HUVEC apoptosis that prompting compound (I) can suppress ox-LDL to induce.The apoptosis rate of compound (I) various dose group reduces gradually along with dosage raises, and between each group, difference has statistical significance (P<0.01).The results are shown in Table 4 (VSox-LDLgroup
#p<0.01).The protection of ecs effect of prompting compound (I) has dose-effect relationship within the specific limits.
Conclusion, this research adopts mtt assay to detect and finds that compound (I) can significantly improve the cell viability of the Human umbilical vein endothelial cells of ox-LDL damage, illustrates that it has the effect of the HUVEC damage suppressing ox-LDL induction.The two staining for flow cell art of further employing AnnexinV-FITC, PI detects apoptosis, and research finds that each concentration group compound (I) all significantly can reduce the HUVEC apoptosis of ox-LDL induction, and effect is dose-dependently.Prompting compound (I) realizes Endothelium Protective effect by suppressing the HUVEC apoptosis of ox-LDL induction.
Table 1 different concns compound (I) on the impact of HUVEC cell viability (
n=6)
| Group | Cell viability (%) |
| Control (control group) | 100.14±3.82 |
| Compound (I) 5 μ g/mL | 100.45±9.67 |
| Compound (I) 10 μ g/mL | 101.22±10.57 |
| Compound (I) 20 μ g/mL | 92.77±8.54 |
| Compound (I) 40 μ g/mL | 48.92±5.62 # |
| Compound (I) 60 μ g/mL | 32.79±2.54 # |
| Compound (I) 80 μ g/mL | 30.21±2.28 # |
| Fvalue | 298.51 |
| Pvalue | <0.01 |
Table 2ox-LDL on the impact of HUVEC cell viability (
n=6)
| Group | Cell viability (%) |
| Control (control group) | 100.02±5.01 |
| ox-LDL10μg/mL | 104.52±4.01 |
| ox-LDL20μg/mL | 93.484.77 * |
| ox-LDL40μg/mL | 86.96±5.78 # |
| ox-LDL60μg/mL | 64.22±3.58 # |
| ox-LDL80μg/mL | 57.21±2.54 # |
| Fvalue | 116.248 |
| Pvalue | <0.001 |
The different immunomodulator compounds of table 3 (I) to ox-LDL induce HUVEC damage intervention effect (
n=6)
| Group | Cell viability (%) |
| Control (control group) | 100.01±3.74 # |
| ox-LDL50μg/mL | 73.92±7.04 |
| Ox-LDL50 μ g/mL+ compound (I) 5 μ g/mL | 82.59±4.80 # |
| Ox-LDL50 μ g/mL+ compound (I) 10 μ g/mL | 90.01±3.70 # |
| Ox-LDL50 μ g/mL+ compound (I) 20 μ g/mL | 93.61±2.11 # |
| Fvalue | 29.15 |
| Pvalue | <0.001 |
Table 4 various dose compound (I) to ox-LDL induce HUVEC apoptosis effect (
n=3)
| Group | Apoptosis rate (%) |
| Control (control group) | 1.84±0.05 # |
| ox-LDL50μg/mL | 9.87±0.40 |
| Ox-LDL50 μ g/mL+ compound (I) 5 μ g/mL | 4.65±0.56 # |
| Ox-LDL50 μ g/mL+ compound (I) 10 μ g/mL | 3.67±0.23 # |
| Ox-LDL50 μ g/mL+ compound (I) 20 μ g/mL | 2.51±0.17 # |
| Fvalue | 274.678 |
| Pvalue | <0.001 |
Embodiment 3
The preparation of tablet: by embodiment 1 method first obtained compound (I), and the salt utilizing organic acid to make as tartrate or citric acid or formic acid or oxalic acid etc., mineral acid example hydrochloric acid or sulfuric acid or phosphoric acid, vehicle is added, pelletizing press sheet than the ratio for 1:7 in itself and excipient weight.
Embodiment 4
Prepared by oral liquid: by embodiment 1 method first obtained compound (I), and the salt utilizing organic acid to make as tartrate or citric acid or formic acid or oxalic acid etc., mineral acid example hydrochloric acid or sulfuric acid or phosphoric acid, oral liquid method for making makes oral liquid routinely.
Embodiment 5
The preparation of capsule or granule: by embodiment 1 method first obtained compound (I), and the salt utilizing organic acid to make as tartrate or citric acid or formic acid or oxalic acid etc., mineral acid example hydrochloric acid or sulfuric acid or phosphoric acid, add vehicle in itself and excipient weight than the ratio for 1:7, make capsule or granule.
Embodiment 6
The preparation of injection liquid: by embodiment 1 method first obtained compound (I), and the salt utilizing organic acid to make as tartrate or citric acid or formic acid or oxalic acid etc., mineral acid example hydrochloric acid or sulfuric acid or phosphoric acid, inject with water routinely, essence filter, injection liquid is made in embedding sterilizing.
Embodiment 7
The preparation of aseptic powder injection: by embodiment 1 method first obtained compound (I), and the salt utilizing organic acid to make as tartrate or citric acid or formic acid or oxalic acid etc., mineral acid example hydrochloric acid or sulfuric acid or phosphoric acid, be dissolved in sterile water for injection, stirring makes molten, filter with aseptic suction funnel, aseptic essence filter again, be sub-packed in ampoule, after frozen drying, aseptic sealing by fusing obtains powder injection.
The effect of above-described embodiment is essentiality content of the present invention is described, but does not limit protection scope of the present invention with this.Those of ordinary skill in the art should be appreciated that and can modify to technical scheme of the present invention or equivalent replacement, and does not depart from essence and the protection domain of technical solution of the present invention.
Claims (7)
1. there is the compound (I) of following structural formula,
2. the preparation method of compound according to claim 1 (I), it is characterized in that comprising following operation steps: the dry mature fruit of Szechwan Chinaberry Fruit is pulverized by (a), extract with 70 ~ 80% alcohol heat reflux, united extraction liquid, be concentrated into without alcohol taste, use sherwood oil, ethyl acetate and water saturated n-butanol extraction successively, obtain petroleum ether extract, acetic acid ethyl ester extract and n-butyl alcohol extract respectively; Acetic acid ethyl ester extract macroporous resin removal of impurities in (b) step (a), first use 10% ethanol elution, 8 column volumes, then use 80% ethanol elution, 10 column volumes, collect 80% ethanol eluate, concentrating under reduced pressure obtains 80% ethanol elution thing medicinal extract; C in () step (b), 80% ethanol elution medicinal extract purification on normal-phase silica gel is separated, obtain 5 components successively with the methylene chloride-methanol gradient elution that volume ratio is 75:1,45:1,25:1,15:1 and 1:1; D in () step (c), component 4 is separated further by purification on normal-phase silica gel, obtain 3 components successively with the methylene chloride-methanol gradient elution that volume ratio is 20:1,15:1 and 8:1; E in () step (d), component 2 reverse phase silica gel of octadecylsilane bonding is separated, with the methanol aqueous solution isocratic elution that concentration expressed in percentage by volume is 70%, collect 10 ~ 12 column volume elutriants, elutriant concentrating under reduced pressure obtains pure compound (I).
3. the preparation method of compound according to claim 2 (I), is characterized in that: described macroporous resin is D101 macroporous adsorbent resin.
4. the preparation method of compound according to claim 2 (I), is characterized in that: it is 75% that described alcohol heat reflux extracts the alcohol concn adopted.
5. a pharmaceutical composition, is characterized in that: the compound according to claim 1 (I) wherein containing treatment significant quantity and pharmaceutically acceptable carrier.
6. the application of compound according to claim 1 (I) in the medicine preparing endotheliocyte protection.
7. the application of pharmaceutical composition according to claim 5 in the medicine preparing endotheliocyte protection.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105254492A (en) * | 2015-11-25 | 2016-01-20 | 杨飞杰 | Novel triterpenoid for liver protection and preparation method thereof |
| CN105859671A (en) * | 2016-04-17 | 2016-08-17 | 温州统益生物医药科技有限公司 | Sesquiterpenoids for medicine and preparation method thereof |
| CN105853407A (en) * | 2016-04-17 | 2016-08-17 | 温州统益生物医药科技有限公司 | Application of sesquiterpenoid in preparing medicine for protecting endothelial cells |
| CN105968079A (en) * | 2016-05-16 | 2016-09-28 | 苏州毕诺佳医药技术有限公司 | Novel diphenyl ethylene type compound, and preparation method and medical purpose thereof |
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| CN102940687A (en) * | 2011-12-12 | 2013-02-27 | 苏州大学 | Szechwan Chinaberry fruit extract and application thereof |
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| CN102940687A (en) * | 2011-12-12 | 2013-02-27 | 苏州大学 | Szechwan Chinaberry fruit extract and application thereof |
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| CHUN-MAO YUAN ET AL.: "Bioactive Limonoid and Triterpenoid Constituents of Turraea Pubescens", 《JOURNAL OF NATURAL PRODUCTS》 * |
| 刘彬: "丹酚酸B对ox-LDL诱导人脐静脉内皮细胞凋亡干预作用的研究", 《中国优秀硕士学位论文全文数据库 医药卫生科技辑》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105254492A (en) * | 2015-11-25 | 2016-01-20 | 杨飞杰 | Novel triterpenoid for liver protection and preparation method thereof |
| CN105859671A (en) * | 2016-04-17 | 2016-08-17 | 温州统益生物医药科技有限公司 | Sesquiterpenoids for medicine and preparation method thereof |
| CN105853407A (en) * | 2016-04-17 | 2016-08-17 | 温州统益生物医药科技有限公司 | Application of sesquiterpenoid in preparing medicine for protecting endothelial cells |
| CN105968079A (en) * | 2016-05-16 | 2016-09-28 | 苏州毕诺佳医药技术有限公司 | Novel diphenyl ethylene type compound, and preparation method and medical purpose thereof |
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