CN108904490A - Application of the flavanone derivative in the drug for preparing anti-hepatic cell fattydegeneration in penthorum chinense pursh - Google Patents
Application of the flavanone derivative in the drug for preparing anti-hepatic cell fattydegeneration in penthorum chinense pursh Download PDFInfo
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Abstract
The present invention relates to application of the flavanone derivative in penthorum chinense pursh in the drug for preparing anti-hepatic cell fattydegeneration.The structural formula of the flavanone derivative is as shown in formula I -1, I -2 or I -3.The present invention has studied the effect of the anti-hepatic cell fattydegeneration of 3 flavanones in penthorum chinense pursh medicinal extract.Penthorum chinense pursh medicinal extract is divided by 5 components using MCI column, the 3 characteristic flavanones chosen in component V obtain pinocembrin (PCB), pinocembrin -7-O- β-D-Glucose glycosides (PCBG), 5- methoxyl group-pinocembrin -7-O- β-D-Glucose glycosides (MPG), investigate the horizontal influence of PCBG, PCB, MPG to cell activity, lipid content, TC, TG, ALT, AST, MDA, SOD and GSH-Px using free fatty acid induction HepG2 cellular damage model.The result shows that 3 feature flavanones all have the activity of anti-hepatic cell fattydegeneration in penthorum chinense pursh medicinal extract.
Description
Technical field
The invention belongs to field of medicaments, and in particular to flavanone derivative is preparing anti-liver cell fat change in penthorum chinense pursh
Application in the drug of property.
Background technique
The whole world hair of nonalcoholic fatty liver (Nonalcoholic Fatty Liver Disease, NAFLD) in recent years
Sick rate increases year by year, and with the improvement of people ' s living standards with the variation of dietary structure, NAFLD has become common chronic in China
One of hepatopathy, the disease incidence in China big and medium-sized cities have reached 15%-25%.Wherein 10% patient develops into nonalcoholic fatty liver
Hepatitis (nonalcoholic steatohepatitis, NASH), nearly 30% patient can develop as cirrhosis or even some diseases
People can develop as liver cancer.Generally believe that NAFLD is that a kind of heredity-environment-metabolism-stress be diseases related at present, with fat, sugar
It is closely related to urinate disease, hyperlipidemia, metabolic syndrome and insulin resistance (insulin resistance, IR).To NAFLD's
Treat mainly according to its pathogenesis be directed to disease different pathological link, by improve insulin resistance, it is anti-oxidant, anti-inflammatory,
Liver protection and lipid-loweringing etc. promote intrahepatic fat and inflammation to disappear, and prevent hepatocyte death and fibrosis.It is confirmed by research, perhaps
More natural drugs have the function of anti-oxidant, anti-inflammatory or increase insulin sensitivity, and to improving, NAFLD is significant in efficacy, therefore
Development effective ingredient in Chinese and ingredient can fully demonstrate traditional Chinese medicine for the research for the treatment of nonalcoholic fatty liver and treat
Unique advantage in terms of NAFLD is aided with stringent quality control, will be the following original new drug in conjunction with long-term clinical practice experience
Development Trend.
Penthorum chinense pursh (Penthorum chinense Pursh.) also known as Chinese penthorum herb, adina pull root vegetables etc., are kiss-mes
Section (Saxifragaceae) penthorum plant pulls the aerial part of root vegetables, is Miao ethnic group's conventional medicament, is distributed mainly on China four
One band of river and Yunnan-Guizhou, with Sichuan Gulin County for the genuine place of production.Its is warm-natured, sweet in flavor, and there is clearing heat and detoxicating, activating microcirculation and removing stasis medicinal, Li Shui to disappear
The effect of swollen, removing jaundice, calming the liver.Using penthorum chinense pursh as penthorum chinense pursh medicine materical crude slice, patent medicine GANSU KELI made of raw material, liver Soviet Union capsule etc., tool
Have drop enzyme, liver protection, removing jaundice, invigorating the spleen function.The chemical components such as isolated flavonoids, terpene from penthorum chinense pursh at present.It catches up with
The hepatoprotective effect of yellow grass is mainly manifested in the sides such as protective effect, anti-hepatic fibrosis and treatment hepatitis B to alcoholic fatty liver
Face, GANSU KELI are clinically widely used in the symptom for improving chronic hepatitis B and liver fibrosis.And to non-alcoholic rouge
The intervention effect research report of fat liver is less.
Summary of the invention
The object of the present invention is to provide the new medicine uses of flavanone derivative.
The new application of flavanone derivative provided by the present invention is that it is preparing the application in following product：
1) drug of anti-hepatic cell fattydegeneration；
2) drug of the accumulation of lipid of liver cell caused by free fatty acid is reduced；
3) drug of the oxidation resistance of liver cell is improved；
4) enhancing liver cell resists the drug of lesion capability；
5) prevent and/or treat the drug of nonalcoholic fatty liver.
The flavanone derivative, structural formula is as shown in formula I -1, I -2 or I -3：
The entitled pinocembrin of compound shown in formula I -1, abbreviation PCB；
Entitled pinocembrin -7-O- β-D-Glucose glycosides of compound shown in formula I -2, abbreviation PCBG；
The entitled 5- methoxyl group of compound shown in formula I-3-pinocembrin-7-O- β-D-Glucose glycosides, abbreviation MPG.
Compound shown in above-mentioned formula I -1, I -2 or I -3 can be extracted from penthorum chinense pursh to be obtained.
The extraction includes：Penthorum chinense pursh medicinal extract is separated by water extract part, component II, component III, group using MCI column
Divide IV and component V, further separation component V, obtains pinocembrin, pinocembrin -7-O- β-D-Glucose glycosides and 5- methoxyl group-Himalayan pine
Element -7-O- β-D-Glucose glycosides.
The penthorum chinense pursh medicinal extract is purchased from Sichuan Gulin Gansu Medicine Co., Ltd, can also be by the side that includes the following steps
Method is prepared：
Penthorum chinense pursh aerial part is added water to cook, collecting decoction, filtered, it is 1.15-1.18 that filtrate, which is condensed into relative density,
The clear cream of (60-65 DEG C), it is cooling, add ethyl alcohol to make alcohol content up to 60%, stir, stands, filtration precipitates 60% ethanol washing,
Merge washing lotion and filtrate, recycle ethyl alcohol and be condensed into the thick paste that relative density is 1.30-1.32 to get.
The water extract part, component II, component III, component IV and component V are obtained by operations described below respectively：Using
Penthorum chinense pursh medicinal extract is successively used water, the ethanol solution of volumetric concentration 20%, the ethanol solution of volumetric concentration 40%, volume by MCI column
The ethanol solution of concentration 60% and the ethanol solution of volumetric concentration 85% are eluted, and collect eluent respectively, obtain water extraction
Position, component II, component III, component IV and component V.
The component V by operations described below further compound shown in isolated formula I -1, compound shown in formula I -2 and
Compound shown in formula I -3：Fr.30 fraction in component V is recrystallizing repeatedly so as to obtain PCBG through methanol；By Fr.32 and Fr.35 with partly
MPG and PCB is made in preparative high-performance liquid chromatographic；
Wherein, preparing chromatographic condition is：Chromatographic column：Hibar 250-10column (10 × 250mm, 5 μm)；Mobile phase：
A- acetonitrile, B- water；Gradient elution：0-10min 20%A, 10-50min 20%-50%A, 50-60min50%A；Flow velocity：
3mL/min。
The above-mentioned component V isolated by penthorum chinense pursh medicinal extract and its prepare in following products application also belong to the present invention
Protection scope：
1) drug of anti-hepatic cell fattydegeneration；
2) drug of the accumulation of lipid of liver cell caused by free fatty acid is reduced；
3) drug of the oxidation resistance of liver cell is improved；
4) enhancing liver cell resists the drug of lesion capability；
5) prevent and/or treat the drug of nonalcoholic fatty liver.
The present invention also provides the rouge of liver cell caused by a kind of drug of anti-hepatic cell fattydegeneration, reduction free fatty acid
The drug of matter accumulation, the drug of the oxidation resistance of raising liver cell, enhancing liver cell resist the drug of lesion capability, and/or
The drug of prevention and/or treatment nonalcoholic fatty liver, the drug include flavanone shown in above-mentioned formula I -1, I -2 or I -3
Derivative.
The present invention has studied the effect of the anti-hepatic cell fattydegeneration of 3 flavanones in penthorum chinense pursh medicinal extract.Using MCI column
Penthorum chinense pursh medicinal extract is divided into 5 components, the 3 characteristic flavanones chosen in component V obtain pinocembrin, pinocembrin -7-O-
β-D-Glucose glycosides, 5- methoxyl group-pinocembrin -7-O- β-D-Glucose glycosides induce HepG2 cellular damage using free fatty acid
Model investigate PCBG, PCB, MPG to cell activity, lipid content, TC, TG, ALT, AST, MDA, SOD and GSH-Px it is horizontal
It influences.The result shows that 3 feature flavanones all have the activity of anti-hepatic cell fattydegeneration in penthorum chinense pursh medicinal extract.
Detailed description of the invention
Fig. 1 is the HPLC chromatogram that penthorum chinense pursh medicinal extract and different concentration ethanol elute position, wherein：A- penthorum chinense pursh medicinal extract；
B- component II；C- component III；D- component IV；E- component V.
Fig. 2 is cell survival rate of the penthorum chinense pursh each component to the FFA HepG2 cell model induced, wherein：A- component II；
B- component III；C- component IV；D- component V.
Fig. 3 is the HepG2 cell oil red O stain mirror following figure (400 ×), wherein：A：Normal group；B：Model group；C：FFA-II；
D：FFA-III；E：FFA-IV；F：FFA-V.
Fig. 4 is that oil red O stain measures HepG2 cytolipin content, compared with normal group, * * p<0.01；With model group ratio
Compared with,Δp<0.05,△△p<0.01。
Fig. 5 is that (A) TG, (B) TC, (C) AST and (D) ALT are horizontal in HepG2 cell, (A) TG, (B) in HepG2 cell
TC, (C) AST and (D) ALT are horizontal.
Fig. 6 is MDA content in HepG2 cell, compared with blank group, * * p<0.01；Compared with model group,Δp<0.05,△△
p<0.01。
Fig. 7 is the cell inhibitory rate of various concentration PCBG, PCB and MPG to the FFA HepG2 cell model induced.
Fig. 8 is the oil red O stain difference group mirror following figure (400 ×) and lipid content.
Fig. 9 is that (A) TC, (B) TG, (C) ALT and (D) AST are horizontal in HepG2 cell, is as a result shown with mean ± SD,##P<
0.01vs control,*P<0.05vs model,**P<0.01vs model。
Figure 10 is that (A) SOD, (B) MDA and (C) GSH-Px are horizontal in HepG2 cell, is as a result shown with mean ± SD,#P<
0.05vs control,##P<0.01vs control,*P<0.05vs model,**P<0.01vsmodel。
Specific embodiment
The present invention will be described below by way of specific embodiments, but the present invention is not limited thereto.
Experimental method used in following embodiments is conventional method unless otherwise specified；Institute in following embodiments
Reagent, biomaterial etc., are commercially available unless otherwise specified.
1, experimental material
1.1 reagent
MCI GEL CHP/CHPMG resin (polystyrene-based reversed-phase resin), Mitsubishi's fine chemistry；Acetonitrile (chromatographically pure),
FISHER Reagent Company of the U.S.；95% ethyl alcohol, Sinopharm Chemical Reagent Co., Ltd.；Distilled water, Wahaha Pure Water；Tire ox
Serum (FBS), DMEM high glucose medium, PBS solution (pH 7.4) and 0.25% trypsase contain EDTA, Gibco company of the U.S.；
MTT, oleic acid (OA), palmitinic acid (PA), bovine serum albumin(BSA) (BSA), cell grade dimethyl sulfoxide (DMSO), U.S. Sigma are public
Department；BCA protein content detection kit, KeyGEN BioTECH company；Oil red O stain kit, triglycerides (TG) test
Box, total cholesterol (TC) testing cassete, glutamic-pyruvic transaminase (ALT) testing cassete, glutamic-oxalacetic transaminease (AST) testing cassete, malonaldehyde (MDA)
Content measuring box, superoxide dismutase (SOD) assay kit, glutathione peroxidase (GSH-Px) measure reagent
Biotech firm is built up in box, Nanjing；Ultrapure water, the preparation of laboratory Mili-Q water purification machine；Other reagents are that analysis is pure.
1.2 experimental cell
The liver cancer cell lines HepG2 cell of people is purchased from American Type Culture Collection (ATCC).
1.3 laboratory sample
Penthorum chinense pursh medicinal extract is purchased from Sichuan Gulin Gansu Medicine Co., Ltd, is accredited as and is caught up with by the said firm senior engineer Zhang Li
The extract of the aerial part of yellow grass Penthorum chinense Pursh., preparation method are：Penthorum chinense pursh 1000g is taken, is cut
It is broken, it adds water to cook three times, 2 hours every time, collecting decoction, filters, it is 1.15-1.18 (60-65 that filtrate, which is condensed into relative density,
DEG C) clear cream, it is cooling, add ethyl alcohol to make alcohol content up to 60%, stir, stand, filtration, precipitating with 60% ethanol washing three times, conjunction
And washing lotion and filtrate, recycling ethyl alcohol are simultaneously condensed into the thick paste that relative density is 1.30-1.32.Medicinal extract is existing in the Capital University of Medical Sciences
Basic scientific research building bioactive components research laboratory, number are:111138.
Pinocembrin -7-O- β-D-Glucose glycosides (PCBG), pinocembrin (PCB), 5- methoxyl group-Portugal pinocembrin -7-O- β-D-
Polyglycoside (MPG) (traditional Chinese medicine institute of Capital University of Medical Sciences self-control.Through UV,1H-NMR、13C-NMR, IR, MS, which identify, determines its chemistry
Structure is detected at 280nm wavelength using HPLC-UV method, 98%) peak area normalization calculated purity is all larger than.
1.4 laboratory apparatus
The ultra-clean Biohazard Safety Equipment of ESCD AC2-4S1 (Thermo company of the U.S.), 371 type carbon dioxide constant incubators (beauty
Thermo company of state), MSL-3020 autoclave (Japanese SANYO company), XDTD-8222 constant temperature blast drying oven (Shanghai
Jing Hong experimental facilities Co., Ltd), W-01B digital display thermostat water bath (Jiangsu Jin Yi instrument Science and Technology Ltd.), blood count
Plate (Shanghai refinement biochemical corp), Transferpette-S single track is adjustable liquid-transfering gun (0.1-1 μ L), Transferpette-S are mono-
Road is adjustable liquid-transfering gun (0.5-10 μ L), Transferpette-S single track is adjustable liquid-transfering gun (10-100 μ L), Transferpette-
S single track is adjustable liquid-transfering gun (20-200 μ L), Transferpette-S single track is adjustable liquid-transfering gun (100-1000 μ L) (German Brand
Company), 5100-0001 type program temperature reduction box (Thermo company of the U.S.), tri- mesh inverted phase contrast microscope of LWD300-38LTI (on
Hai Cewei photoelectricity company), 384 all-wave length microplate reader of SpectraMax Plus (Molecular Devices company of the U.S.),
0.22 μm of syringe needle filter of Millex GV (Millipore company of the U.S.), ECLIPSE 80i advanced studies type microscope (Japan
Nikon company), ten a ten thousandth electronic analytical balance of ME235S (Sai Duolisi scientific instrument Co., Ltd), KQ5200DV number
It controls ultrasonic cleaner (Kunshan Ultrasonic Instruments Co., Ltd.), 25mL Tissue Culture Flask, 96 well culture plates, 6 well culture plates (beauty
Corning company of state), 15mL centrifuge tube, 50mL centrifuge tube, 2mL cryopreservation tube (Corning company of the U.S.), TDL-60B it is desk-top from
Scheming (Anting Scientific Instrument Factory, Shanghai), BCD-254 refrigerator (German SIEMENS company), ULT-2186-4-v49 ultralow temperature ice
Case (Thermo company of the U.S.), Primo Vert inverted microscope (German Carl Zeiss company).
2, experimental method and result
2.1 effective fraction
2.1.1 the preparation at penthorum chinense pursh medicinal extract difference elution position
Penthorum chinense pursh medicinal extract is dissolved with 50% ethanol solution of volumetric concentration, water, 20% second of volumetric concentration is respectively adopted through MCI column
Alcoholic solution, 40% ethanol solution of volumetric concentration, 60% ethanol solution of volumetric concentration and 85% ethanol solution of volumetric concentration are washed
It is de-, obtain 20% elution position (component II), 40% elution position (component III), 60% elution position (component IV) and 85%
It elutes position (component V).
With high effective liquid chromatography for measuring penthorum chinense pursh medicinal extract, component II, component III, component IV, component V, such as Fig. 1.Chromatography
Condition：Using octadecylsilane chemically bonded silica as filler (Agilent SB C18 column, column length 25cm, internal diameter 4.6mm,
Partial size is 5 μm)；Using 0.2% aqueous formic acid as mobile phase A, using acetonitrile as Mobile phase B, gradient 0-65min, 10%-
50%B；30 DEG C of column temperature；Flow velocity 1.0mL/min；Detection wavelength is 280nm.
2.1.2 the external activity screening of penthorum chinense pursh medicinal extract each component
Component II, component III, component IV and component V 6.8mg, 12mg, 13.2mg and 16mg are weighed respectively, with 100 μ L
DMSO is dissolved as the mother liquor of 68mg/mL, 120mg/mL, 132mg/mL, 160mg/mL, using preceding complete with the DMEM containing 0.5%BSA
Full culture medium is diluted to using concentration, crosses 0.22 μm of filter membrane.
The HepG2 cell of logarithmic growth phase, with 5 × 104The density in the hole cell/ is inoculated in 96 orifice plates, culture for 24 hours to
After the adherent stable growth of cell, 0.8mM FFA modeling is added, while giving the penthorum chinense pursh each component of cell various concentration respectively,
Making final concentration is respectively 8.5,17,34,85,170,340 μ g/mL of component II, 10,50,100,300,600 μ g/ of component III
10,50,100,200 μ g/mL of mL, component IV 33,66,165,330,660 μ g/mL, component V.Blank control group is set simultaneously
And model group, every group is all provided with 6 parallel multiple holes, 37 DEG C, 5%CO2After continuing culture for 24 hours, every hole is added 10 μ L 5mg/mL's
MTT solution mixes, in 37 DEG C, 5%CO2Continue after cultivating 4h, inhale and abandon culture medium in hole, 100 μ L DMSO dissolution is added, mixes
It is even, it (is indicated with MTT colorimetric method for determining cell proliferative conditions with absorbance OD value under 490nm).As a result as shown in Figure 2.Finally set
Setting administration concentration is respectively：17 μ g/mL of component II；300 μ g/mL of component III；66 μ g/mL of component IV；100 μ g/ of component V
mL。
The sterile cover slips of 1.8 × 1.8cm are put into 6 orifice plates, cell climbing sheet is made.The HepG2 of logarithmic growth phase
Cell, with 2 × 105The density in the hole cell/ is inoculated in the 6 orifice plates equipped with coverslip, and culture is for 24 hours to the adherent stable growth of cell
Afterwards, the DMEM complete medium of 0.5%BSA is added in control group, and 0.8mM FFA is added in model group, and administration group is separately added into 0.8mM
17 μ g/mL of FFA and component II, 300 μ g/mL of component III, 66 μ g/mL of component IV, 100 μ g/mL of component V, 37 DEG C, 5%
CO2Continue culture for 24 hours.It is operated according to the specification of oil red O stain kit, the feelings of microscopic observation cell red fat drips after mounting
Condition, each group lipid profile are as shown in Figure 3.It is washed 2-3 times with PBS, every hole 0.5mL isopropanol dissolves fat drips, 5min is vibrated, wait shake
After even, (indicating with absorbance OD value) is detected under microplate reader 510nm, with model group absorbance for 100%, each group lipid content knot
Fruit sees Fig. 4.
The HepG2 cell of logarithmic growth phase, with 1 × 106The density in the hole cell/ is inoculated in the culture dish that diameter is 10cm
In, culture is for 24 hours after the adherent stable growth of cell, and the DMEM complete medium of 0.5%BSA is added in control group, and model group is added
0.8mM FFA, administration group are separately added into 17 μ g/mL of 0.8mM FFA and component II, 300 μ g/mL of component III, 66 μ of component IV
G/mL, 100 μ g/mL of component V, 37 DEG C, 5%CO2Continue culture for 24 hours.Cell is collected, is dissolved with 1mL PBS buffer solution, ultrasound
Crush method ice bath is 3 times broken, each 10s.BCA method measures the protein content of cell, according to triglycerides (TG) testing cassete, total gallbladder
Sterol (TC) testing cassete, glutamic-pyruvic transaminase (ALT) testing cassete, glutamic-oxalacetic transaminease (AST) testing cassete and malonaldehyde (MDA) kit
Method is measured.
0.8mM FFA induce HepG2 cell for 24 hours after, TC and TG content obviously increases in cell suspension, and ALT and AST are horizontal
Also significantly raised, compared with Normal group, difference has statistical significance (p<0.01)；Give penthorum chinense pursh each component intervention
Each group, TC and TG content is declined compared with model group in cell suspension, more significant with component II and component III administration group
(p<0.05)；ALT and AST level is declined (p compared with model group in cell suspension<0.01), wherein with component IV and component V
Administration group is the most significant.As a result see Fig. 5.
0.8mM FFA induce HepG2 cell for 24 hours after, MDA content obviously increases in cell suspension, with Normal group phase
Compare, difference has statistical significance (p<0.01)；After giving the intervention of penthorum chinense pursh each component, MDA content in group of cells suspension
Declined compared with model group, with the more significant (p of component V administration group<0.01).See Fig. 6.
Fr.30 fraction in component V is recrystallizing repeatedly so as to obtain PCBG through methanol；Fr.32 and Fr.35 is prepared efficiently with half
MPG and PCB is made in liquid chromatogram.Preparing chromatographic condition is chromatographic column：Hibar 250-10column (10 × 250mm, 5 μm)；
Mobile phase：A- acetonitrile, B- water；Gradient elution：0-10min 20%A, 10-50min20%-50%A, 50-60min 50%A；
Flow velocity：3mL/min.
5- methoxyl group-pinocembrin -7-O- β-D-Glucose glycosides (MPG)：
White, needle-shaped crystals (methanol-acetone), ESI-MS m/z 433.1488 [M+H]+,1H NMR(600MHz,CD3OD)
δ:5.47 (1H, dd, J=12.6,3.0Hz, H-2), 3.04 (1H, dd, J=16.8,12.6Hz, H-3 α), 2.77 (1H, dd, J
=16.8,3.0Hz, H-3 β), 6.38 (1H, d, J=1.8Hz, H-6), 6.41 (1H, d, J=2.4Hz, H-8), 7.49 (2H, d,
J=7.8Hz, H-2', 6'), 7.39 (3H, m, H-3', 4', 5'), 5.01 (1H, d, J=7.8Hz, H-1 "), 3.20-4.00
(5H, m, H-2 ", 3 ", 4 ", 5 ", 6 "), 3.87 (3H, s, MeO)；13C NMR(150MHz,CD3OD)δ:80.6(C-2),46.7
(C-3),192.2(C-4),164.0(C-5),98.3(C-6),166.0(C-7),95.6(C-8),166.4(C-9),107.8
(C-10),140.6(C-1'),127.5(C-2',6'),129.8(3',4',5'),101.8(C-1”),74.9(C-2”),78.1
(C-3 "), 71.6 (C-4 "), 78.7 (C-5 "), 62.7 (C-6 "), 56.7 (MeO)
Pinocembrin -7-O- β-D-Glucose glycosides (PCBG):
White, needle-shaped crystals (methanol-acetone), ESI-MS m/z 417 [M-H]-,1H NMR(500MHz,DMSO-d6)δ:
5.66 (1H, dd, J=12.6,3.0Hz, H-2), 3.15 (1H, dd, J=16.8,12.6Hz, H-3 α), 2.86 (1H, dd, J=
16.8,3.0Hz, H-3 β), 6.17 (1H, d, J=2.4Hz, H-6), 6.21 (1H, d, J=2.4Hz, H-8), 7.54 (2H, d, J
=8.4Hz, H-2', 6'), 7.43 (3H, m, H-3', 4', 5'), 4.98 (1H, d, J=9.0Hz, H-1 "), 3.70 (5H, m, H-
2",3",4",5",6")；13C NMR(125MHz,DMSO-d6)δ:79.1(C-2),42.7(C-3),197.3(C-4),163.0
(C-5),97.1(C-6),165.8(C-7),96.0(C-8),163.4(C-9),103.8(C-10),139.0(C-1'),127.2
(C-2',6'),129.1(3',4',5'),100.1(C-1”),73.5(C-2”),76.8(C-3”),70.0(C-4”),77.5
(C-5”),61.0(C-6”).
Pinocembrin (PCB):
White pinniform crystallizes (methanol), ESI-MS m/z 255 [M-H]-,1H NMR(600MHz,MeOD)δ:5.46(1H,
Dd, J=12.6,3.0Hz, H-2), 3.09 (1H, dd, J=16.8,12.6Hz, H-3 α), 2.78 (1H, dd, J=16.8,
3.0Hz, H-3 β), 5.60 (1H, d, J=2.4Hz, H-6), 5.92 (1H, d, J=2.4Hz, H-8), 7.49 (2H, m, H-2',
6'), 7.36~7.41 (3H, m, H-3', 4', 5')；13C NMR(150MHz,MeOD)δ:80.7(C-2),44.4(C-3),
197.5(C-4),164.9(C-5),97.4(C-6),168.6(C-7),96.5(C-8),165.7(C-9),103.6(C-10),
140.6(C-1'),129.9(C-2',6'),129.8(3',4'),127.5(C-5')。
The anti-hepatic cell fattydegeneration activity of 3.1PCBG, PCB and MPG
The stock solution of PCBG, PCB and MPG are diluted to 1mM with the DMEM complete medium containing 0.5%BSA, with 0.22 μm
Membrane filtration.
The HepG2 cell of logarithmic growth phase, with 5 × 104The density in the hole cell/ is inoculated in 96 orifice plates, culture for 24 hours to
After the adherent stable growth of cell, be separately added into 0.8mM FFA and certain volume 1mM PCBG, PCB and MPG make it is final concentration of
10,50,100,250 μM, every group is all provided with 6 parallel multiple holes, in 37 DEG C, 5%CO2After continuing culture for 24 hours, 10 μ L are added in every hole
The MTT solution of 5mg/mL mixes, in 37 DEG C, 5%CO2Continue after cultivating 4h, inhale and abandon culture medium in hole, 100 μ L DMSO are added
Dissolution is mixed, (is indicated with MTT colorimetric method for determining cell proliferative conditions with absorbance OD value under 490nm), as a result as shown in Figure 7.
High, medium and low administration concentration, which is finally respectively set, is：PCBG 10,1,0.1μM；PCB 100,10,1μM；MPG 100,10,1μM.
The sterile cover slips of 1.8 × 1.8cm are put into 6 orifice plates, cell climbing sheet is made.The HepG2 of logarithmic growth phase
Cell, with 2 × 105The density in the hole cell/ is inoculated in the 6 orifice plates equipped with coverslip, and culture is for 24 hours to the adherent stable growth of cell
Afterwards, the DMEM complete medium of 0.5%BSA is added in control group, and 0.8mM FFA is added in model group, and administration group is separately added into 0.8mM
PCBG, PCB and MPG of FFA and high, medium and low dosage, 37 DEG C, 5%CO2Continue culture for 24 hours.According to oil red O stain kit
Specification operates, after mounting the case where microscopic observation cell red fat drips.It is washed 2-3 times with PBS, every hole is molten with 0.5mL isopropanol
Fat drips are solved, 5min is vibrated, after shaking up, (indicating with absorbance OD value) is detected under microplate reader 510nm, is with model group absorbance
100%, each group lipid content result is shown in Fig. 8.
The HepG2 cell of logarithmic growth phase, with 1 × 106The density in the hole cell/ is inoculated in the culture dish that diameter is 10cm
In, culture is for 24 hours after the adherent stable growth of cell, and the DMEM complete medium of 0.5%BSA is added in control group, and model group is added
0.8mM FFA, administration group are separately added into PCBG, PCB and MPG of 0.8mM FFA and high, medium and low dosage, 37 DEG C, 5%CO2After
Continuous culture is for 24 hours.Cell is collected, is dissolved with 1mL PBS buffer solution, sonioation method ice bath is 3 times broken, each 10s.BCA method is surveyed
The protein content for determining cell is surveyed according to triglycerides (TG) testing cassete, total cholesterol (TC) testing cassete, glutamic-pyruvic transaminase (ALT)
Try box, glutamic-oxalacetic transaminease (AST) testing cassete, malonaldehyde (MDA) kit, superoxide dismutase (SOD) testing cassete and paddy Guang
Sweet peptide peroxidase (GSH-Px) kit method is measured.
0.8mM FFA induce HepG2 cell for 24 hours after, TC and TG content obviously increases in cell suspension, and ALT and AST are horizontal
Also significantly raised, compared with blank control group, difference has statistical significance (p<0.01)；Give senior middle school low concentration PCBG,
The each group that PCB and MPG intervene, TC and TG content is declined compared with model group in cell suspension, with PCBG high middle dose group compared with
For significant (p<0.05)；ALT and AST level is declined (p compared with model group in cell suspension<0.01), as a result there is statistics
Learn difference.See Fig. 9.
0.8mM FFA induce HepG2 cell for 24 hours after, MDA content obviously increases (p in cell suspension<0.01), SOD activity
Decrease (p<0.01), GSH-Px level is significantly reduced (p<0.05), compared with blank control group, difference have statistics
Learn meaning；Senior middle school low concentration PCBG, PCB and MPG each group of intervention are given, MDA content is obvious compared with model group in cell suspension
Decline (p<0.01), the most significant with high dose MPG group；SOD activity is risen (p<0.05), GSH-Px is horizontal significantly raised
(p<0.01), the most significant with high dose MPG group.The result is shown in Figure 10.
It is above-mentioned the experimental results showed that, tri- flavanones of PCB, PCBG, MPG can reduce free-fat in penthorum chinense pursh medicinal extract
The accumulation of lipid of liver cell caused by acid, improves the oxidation resistance of cell, and enhancing liver cell resists lesion capability.
Claims (9)
1. flavanone derivative shown in formula I -1, I -2 or I -3 is preparing the application in following product：
1) drug of anti-hepatic cell fattydegeneration；
2) drug of the accumulation of lipid of liver cell caused by free fatty acid is reduced；
3) drug of the oxidation resistance of liver cell is improved；
4) enhancing liver cell resists the drug of lesion capability；
5) prevent and/or treat the drug of nonalcoholic fatty liver；
2. application according to claim 1, it is characterised in that：Flavanone derivative shown in the formula I -1, I -2 or I -3
It extracts and obtains from penthorum chinense pursh.
3. application according to claim 2, it is characterised in that：The extraction includes：Penthorum chinense pursh medicinal extract is divided using MCI column
Pinocembrin, Himalayan pine are obtained from for water extract part, component II, component III, component IV and component V, further separation component V
Element -7-O- β-D-Glucose glycosides and 5- methoxyl group-pinocembrin -7-O- β-D-Glucose glycosides.
4. application according to claim 3, it is characterised in that：The penthorum chinense pursh medicinal extract passes through the method included the following steps
It is prepared：
Penthorum chinense pursh aerial part is added water to cook, collecting decoction, filtered, it is the clear of 1.15-1.18 that filtrate, which is condensed into relative density,
Cream, it is cooling, add ethyl alcohol to make alcohol content up to 60%, stir, stand, filtration precipitates 60% ethanol washing, merges washing lotion and filter
Liquid, recycle ethyl alcohol and be condensed into relative density be 1.30-1.32 thick paste to get.
5. application according to claim 3 or 4, it is characterised in that：The water extract part, component II, component III, group
Divide IV and component V to pass through operations described below respectively to obtain：Penthorum chinense pursh medicinal extract is successively used by water, volumetric concentration 20% using MCI column
Ethanol solution, the ethanol solution of volumetric concentration 40%, the ethyl alcohol of the ethanol solution of volumetric concentration 60% and volumetric concentration 85% are molten
Liquid is eluted, and collects eluent respectively, obtains water extract part, component II, component III, component IV and component V.
6. the application according to any one of claim 2-5, it is characterised in that：The component V is by operating as follows into one
Walk compound shown in compound shown in isolated formula I -1, compound and formula I -3 shown in formula I -2：By Fr.30 fraction in component V
PCBG is recrystallizing repeatedly so as to obtain through methanol；MPG and PCB is made in half preparative high-performance liquid chromatographic of Fr.32 and Fr.35；
Wherein, preparing chromatographic condition is：Chromatographic column：Hibar 250-10column (10 × 250mm, 5 μm)；Mobile phase：A- second
Nitrile, B- water；Gradient elution：0-10min 20%A, 10-50min 20%-50%A, 50-60min 50%A；Flow velocity：3mL/
min。
7. by component V that penthorum chinense pursh medicinal extract is isolated described in claim 3.
8. the component V isolated by penthorum chinense pursh medicinal extract as claimed in claim 7 is preparing the application in following product：
1) drug of anti-hepatic cell fattydegeneration；
2) drug of the accumulation of lipid of liver cell caused by free fatty acid is reduced；
3) drug of the oxidation resistance of liver cell is improved；
4) enhancing liver cell resists the drug of lesion capability；
5) prevent and/or treat the drug of nonalcoholic fatty liver.
9. a kind of drug of the accumulation of lipid of liver cell caused by drug of anti-hepatic cell fattydegeneration, reduction free fatty acid,
Improve the drug of the oxidation resistance of liver cell, the drug of enhancing liver cell resistance lesion capability or prevention and/or the non-wine for the treatment of
The drug of essence fatty liver, it is characterised in that：The drug includes flavanone shown in claim 1 Chinese style I -1, I -2 or I -3
Derivative；
Or, the drug includes the component V isolated by penthorum chinense pursh medicinal extract as claimed in claim 7.
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Cited By (2)
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| CN110229204A (en) * | 2019-05-29 | 2019-09-13 | 四川大学 | A kind of compound PGHG preparation method treated in Parkinson disease drug |
| CN116270771A (en) * | 2022-09-07 | 2023-06-23 | 浙江中医药大学 | Application of penthorum chinense pursh and extractive thereof |
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| CN110229204B (en) * | 2019-05-29 | 2020-04-17 | 四川大学 | Preparation method of compound PGHG in Parkinson disease protection medicine |
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