CN106031732A - Uses of quercetin glucoside compounds in treatment of hepatic fibrosis - Google Patents
Uses of quercetin glucoside compounds in treatment of hepatic fibrosis Download PDFInfo
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Abstract
The invention relates to the field of medicines, and particularly relates to uses of quercetin glucoside compounds in treatment of hepatic fibrosis. Pharmacodynamic tests prove that the quercetin glucoside compounds have functions of relieving liver injury, hepatic parenchymal cell necrosis and hepatic fibrosis which are caused by CCl4 and have certain liver protecting activity. The quercetin glucoside compounds can also inhibit propagation and activation of HSC cells under inflammation states, thus playing a role of resisting the hepatic fibrosis.
Description
Technical field
The present invention relates to field of medicaments, be specifically related to quercetin glycoside compounds purposes in treatment hepatic fibrosis.
Background technology
Hepatic fibrosis is the persistent inflammation reaction caused by hepatic injury, in liver, quantity and the composition of extracellular matrix change, normal degradation process is suppressed, pile up and increase, too much insoluble matrix is caused to be deposited in liver, cause normal organization to destroy until organ failure, finally develop into liver cirrhosis even hepatocarcinoma.Liver cirrhosis is that liver parenchyma sexually transmitted disease (STD) becomes, and medical circle generally believes more difficult reverse, and hepatic fibrosis is then reversible, and the research therefore reversing hepatic fibrosis has become the hot subject that educational circles pays close attention to.
Numerous studies show, inflammatory reaction chronic in liver is the main inducing causing hepatic fibrosis.Macrophage is the main effects cell of inflammation reaction, and wherein Kupffer cell is topmost resident macrophage in liver.In course of liver damage, Kupffer cell is in addition to directly participating in inflammatory reaction, the multiple rush brotic cells factor can also be secreted (such as TNF-α, IL-1, IL-6, TGF-β, PDGF), stimulate the propagation of hepatic stellate cell (hepatic stellate cells, HSC), migrate and activate, promote extracellular matrix to produce and fibrosis is formed.Wherein, TGF-β is considered as one of factor of maximally effective HSC stimulated cell activation.Additionally, Kupffer cell also discharges substantial amounts of oxygen-derived free radicals (reactive oxygen species, ROS), lasting oxidative stress also can induced activation HSCs, maintain hepatic fibrosis process.
At present, treatment the drug main interferon to be had of hepatic fibrosis, colchicine etc. conventional clinically, but therapeutic effect is limited, and side effect is big.Therefore, market remains a need for the exploitation of effective anti-hepatic fibrosis medicines.
Quercetin belongs to flavone compound, is widely present in multiple medicinal plants and vegetable and fruit.Research shows, Quercetin and glycosides derivative thereof have a pharmacotoxicological effect widely such as antiinflammatory, antioxidation, blood pressure lowering, antidepressant, but so far there are no this compounds application report in preparing Strategies of Anti-fibrosis Therapy medicine.
The structure of Quercetin and glucosides thereof is as follows:
Summary of the invention
The invention discloses the purposes of quercetin glycoside treatment hepatic fibrosis.Quercetin glycoside of the present invention preferred having structure formula:
Wherein R represents-COOH or-CH2X, wherein X represents OH, F, Cl, Br, I, CN, NO2、NH2、CF3、SH、OCH3、OC2H5Or COOH.
R preferably represents-CH2OH, i.e. isoquercitrin (code name IQ).
R further preferably represents-COOH, i.e. Quercetin-3-O-β-D-Glucose aldehydic acid glycosides (code name QB).
R further preferably represents-CH2OCH3.I.e. Quercetin-3-O-β-D-Glucose aldehydic acid methyl ester (code name QC)
R further preferably represents-CH2F.I.e. fluoro isoquercitin glycosides (code name QF).
Part pharmacodynamics test and the result of quercetin glycoside be presented herein below:
One, Effect study in the Rat Liver Fibrosis Model body that CCl4 is caused by quercetin glycoside compounds
Male SD rat 56, body weight 180-200g/, sub-cage rearing, freely drink water, room temperature 20-25 DEG C, illumination 12 hour/day.Random lot is divided into 7 groups, often group 8: 1. normal group (ND)；2. model group (MO)；3. isoquercitrin group (IQ, 25mg/kg/d)；4. Quercetin-3-O-β-D-Glucose aldehydic acid glycosides group (QB, 25mg/kg/d)；5. Quercetin group (Q, 50mg/kg/d)；6. Quercetin-3-O-β-D-Glucose aldehydic acid methyl ester: (QC, 25mg/kg/d)；7. fluoro isoquercitin glycosides: (QF, 25mg/kg/d).Normal group gives olive oil solution 3mL/kg subcutaneous injection, 2 times a week, totally 8 weeks；First dose of remaining group gives 40%CCl4 olive oil solution subcutaneous injection by 5ml/kg, gives by 3ml/kg later, 2 times a week, and totally 8 weeks.Every morning identical time point gavage respectively gives relative medicine, adjusts dosage by body weight weekly, is administered 4 weeks.After administration terminates, fasting 16 hours, femoral artery is taken a blood sample, and takes off cervical vertebra and puts to death, takes liver and prepare serum, liver tissue homogenate routinely.Glutamate pyruvate transaminase (ALT) and glutamic oxaloacetic transaminase, GOT (AST) level in detection serum.Active oxygen (reactive oxygen species, ROS) and SOD level in detection liver organization, and kupffer cell and HSC number.
(1) quercetin glycoside compounds is on Serum ALT and the impact of AST level
Table 1. quercetin glycoside compounds is on Serum ALT and the impact of AST level
Compared to normal group,aP<0.5；Compared to model group,bP<0.5。
Result shows: compared with normal group, and model group Serum ALT and AST level significantly raise；Compared with model group, quercetin glycoside compounds QB, IQ, QC and QF all can significantly lower serum alt and AST level, and Quercetin (Q) can significantly lower AST level, but have not significant impact ALT.Result above shows, CCl4 can cause obvious hepatic injury, causes hepatic parenchymal cells downright bad, hepatic fibrosis occurs, and quercetin glycoside compounds then can reverse this effect, have certain liver-protecting activity.
(2) quercetin glycoside compounds is on the impact of Kupffer cell in liver organization
Result is shown in that Fig. 1, Fig. 1 show: compared with normal group, and in model group liver, Kupffer cell number substantially increases, and its mark representation CD68 content is significantly raised；Compared with model group, quercetin glycoside compounds QB, IQ and QC all can substantially reduce the number of liver organization Kupffer cell in pole, lower the expression of CD68, and QF and Q has certain downward effect to the number of Kupffer cell in liver organization, but effect is the most notable.This shows that quercetin glycoside compounds QB, IQ and QC may be improved the inflammatory conditions in liver by regulation and control Kupffer cell, thus played effect of anti hepatic fibrosis.
(3) quercetin glycoside compounds is on the impact of HSC cell in liver
Result is shown in that Fig. 2, Fig. 2 show: compared with normal group, and in model group liver organization, HSC cell quantity substantially increases；Compared with model group, quercetin glycoside compounds IQ, QB and QC all can obviously reduce the number of HSC cell, and the effect such as figure QB group is best, and HSC cell is had no significant effect by Quercetin.This partial results illustrates, in the course of liver damage of CCl4 induction, quercetin glycoside compounds IQ, QB and QC can suppress the propagation of HSC cell.
(4) quercetin glycoside compounds is on the impact of oxidative and anti-oxidative index in liver organization
Result is shown in that Fig. 3, Fig. 3 show: compared with normal group, and in model group liver, oxidoreduction level is unbalance, and ROS produces and significantly increases, and SOD enzyme activity is decreased obviously；Compared with model group, quercetin glycoside compounds IQ, QB and QC all can substantially reduce the content of ROS in liver, increased SOD vigor, and the oxidation antioxidation level in liver is then had no significant effect by Q.This partial results shows, quercetin glycoside compounds IQ, QB and QC can alleviate the oxidative damage of liver, reduces the generation of ROS, improves SOD vigor, has hepatoprotective effect.
Two, the Kupffer cell that LPS is induced by quercetin glycoside compounds studies (1) rat primary HSC cell and the separation and Culture of Kupffer cell with the interaction in vitro of HSC cell co-culture model
Select normal male SD rat, fixation postures, sterilization after anaesthetizing with 10% chloral hydrate (300 μ L/100g), open skin successively, abdominal cavity exposes portal vein and postcava, insert venous catheter needle after opening thoracic cavity postcava breast section to fix with bulldog clamp, cut off portal vein, simultaneously the air in the emptying trocar, to the HEPES buffer of liver perfusion 37 DEG C preheating, flow velocity 30mL/min.After irrigating 15-20 minute liver gradually become canescence at once stop perfusion, use 0.02% calcic (0.06%) collagenase perfusion of 37 DEG C of preheatings instead, flow velocity is 15mL/min, perfusion is stopped immediately when vesicle or slight crack occurs in liver surface wait seeing, carefully take off to put into by liver and the beaker filling HEPES buffer rinses gently one time, then hepatic metastasis to is filled in the beaker of plasma-free DMEM medium, tear the fibrous membrane of liver surface with aseptic nipper, and constantly shake cell.Cell suspension filters with 200 mesh filter screens, takes the cell suspension after filtration and is centrifuged 90 seconds with 50g, makes the HSC cell in liver, kupffer cell separate with hepatocyte.Taking the supernatant, 50g is centrifuged 5 minutes, repeats 1-2 time, abandons precipitation.Take supernatant 200g to be centrifuged 10 minutes, take precipitation with the DMEM culture medium containing 10%FBS resuspended after, 200g is centrifuged 10 minutes again, abandons supernatant, and remaining precipitation is resuspended by the DMEM culture medium containing 10%FBS, adjust concentration be 2 × 105It is seeded in culture bottle, is placed in 37 DEG C, the CO2 incubator of volume fraction 5% is cultivated, within every 2-3 days, changes a not good liquor.
Mentioned above, 50g be centrifuged 90 seconds after isolating hepatocytes obtain supernatant, 4 DEG C, 450g is centrifuged 10 minutes, it is thus achieved that Hepatic nonparenchymal cell.With Percoll two step gradient centrifugation 1000g, 4 DEG C centrifugal carries out the separation of Kupffer cell, purification in 10 minutes.Resuspended by the DMEM culture medium containing 10%FBS, adjusting concentration is 2 × 105Being seeded in 6 orifice plates, be placed in 37 DEG C, volume fraction 5%CO2 incubator is cultivated.After 2 hours, change liquid first, wash away the most adherent cell, often within 2-3 days, change a not good liquor afterwards.
Using trypan blue staining to measure rat primary HSC cell survival rate is 95 ± 2%, and Kupffer cell survival rate is 96 ± 2%, and result shows, its cell survival rate meets the requirement of In vitro culture.
(2) foundation of Kupffer cell (normally)/HSC cell co-culture model
Taking the Kupffer cell that growth conditions is good, after 0.25% trypsinization, adding 1ml density in the lower room of Millicell (micro-pore diameter of middle semipermeable membrane is 0.4 μm) Double layer culture room is 2 × 105Kupffer cell；The most again by 1mlHSC cell with 2 × 105The density of individual/ml adds Millicell upper strata cell.By the DMEM culture medium containing 10%FBS, it is placed in 37 DEG C, cultivates in the CO2 incubator of volume fraction 5%, within every 2 days, change a subculture.After 5-7 days, HSC cell differentiation and maturation, can test.
(3) foundation of Kupffer cell (inflammatory conditions)/HSC cell co-culture model
Taking the Kupffer cell that growth conditions is good, after 0.25% trypsinization, adding 1ml density in the lower room of Millicell (micro-pore diameter of middle semipermeable membrane is 0.4 μm) Double layer culture room is 2 × 105Kupffer cell, add containing the DMEM culture medium culturing of 10%FBS and 100ng/ml lipopolysaccharide after 24 hours, then by 1mlHSC cell with 2 × 105The density of individual/ml adds Millicell upper strata cell.Now culture medium used is all changed to the DMEM culture medium containing 10%FBS, is placed in 37 DEG C, cultivate in the CO2 incubator of volume fraction 5%, within every 2 days, change a subculture.After 5-7 days, HSC cell differentiation and maturation, can test.
(4) experiment packet and administration process:
Normal group 1:HSC single culture；
Normal group 2:Kupffer cell and HSC co-culture of cells (KC+HSC)；
The Kupffer cell of model group: LPS induction and HSC co-culture of cells；
QC group: 50 μMs；IQ group: 50 μMs；QB group: 30 μMs；Q group: 50 μMs；
Use above two co-culture model, put 37 DEG C, after cultivating 24 hours in volume fraction 5%CO2 incubator, inhale the culture medium abandoning lower cell, lower room adds DMEM culture medium 1ml containing various dose medicine, after cultivating 24 hours or 48 hours, HSC cell number, ROS level and TGF-β content in detection system.
5. experimental result
(1) quercetin glycoside compounds is on the impact of HSC cell number in co-culture model
Result is shown in that Fig. 4, Fig. 4 show: compared with normal group 1, when normal Kupffer cell and HSC co-culture of cells 24 are little or when 48 is little, in system, HSC cell number increased, but does not has significant difference；Compared with normal group 2, when the Kupffer cell under the inflammatory conditions using LPS induction and HSC co-culture of cells, either 24 hours or 48 hours, in system, the number of HSC cell the most substantially increased；And compared with model group, no matter quercetin glycoside compounds IQ and QB acts on 24h or 48h all can significantly reduce the quantity of HSC cell in system, and QC effect 24h also can substantially reduce the quantity of HSC cell, but HSC cell is had no significant effect by medicine Q.This partial results shows, quercetin glycoside compounds IQ, QB and QC all can suppress propagation and the activation of HSC cell under inflammatory conditions to a certain extent, thus play the effect of anti-hepatic fibrosis.
(2) quercetin glycoside compounds is on the impact of ROS level in co-culture model
Result is shown in that Fig. 5, Fig. 5 show: compared with normal group 1, and when normal Kupffer cell and HSC co-culture of cells, in system, ROS level slightly raises, but does not has significant difference；Compared with normal group 2, when the Kupffer cell under the inflammatory conditions using LPS induction and HSC co-culture of cells, in system, ROS level significantly raises；And compared with model group, quercetin glycoside compounds QC, IQ and QB all can significantly lower ROS level, wherein the effect with IQ and QB is best (P < 0.001), and ROS level is had no significant effect by medicine Q.This partial results shows, quercetin glycoside compounds QC, IQ and QB can substantially reduce the generation of ROS in co-culture system under inflammatory conditions, the balance of maintenance system oxidative and anti-oxidative state in pole.Therefore, quercetin glycoside compounds QC, IQ and QB are likely to by the activation producing minimizing HSC cell of ROS in inhibition system, thus play the effect of anti-hepatic fibrosis.
(3) quercetin glycoside compounds is on the impact of TGF-β content in co-culture model
Result is shown in that Fig. 6, Fig. 6 show: compared with normal group 1, when normal Kupffer cell and HSC co-culture of cells 24 are little or 48 little constantly, in system, the content of TGF-β somewhat raises, but does not has significant difference；Compared with normal group 2, when the Kupffer cell under the inflammatory conditions using LPS induction and HSC co-culture of cells, either 24 hours or 48 hours, in system, the content of TGF-β the most substantially raised；And compared with model group, no matter quercetin glycoside compounds QC, IQ and QB act on 24h or 48h all can the content of TGF-β, wherein QC and QB effect 48h the most notable (P < 0.01) in substantially downward system.This partial results shows, quercetin glycoside compounds IQ, QB and QC all can reduce the generation promoting the fibrosis factor, the activation of suppression HSC cell by suppressing the release of TGF-β, and the effect of QC and QB is more preferable.But Quercetin Q has no notable biological effect.
Accompanying drawing explanation
Fig. 1. it is that quercetin glycoside compounds is on the impact (note: white arrow show the expression of kupffer cell sign thing CD68) of Kupffer cell in liver
Fig. 2 is that quercetin glycoside compounds is on the impact (in figure, navy blue point shown in black arrow is HSC cell) of HSC cell number in liver
Fig. 3 be quercetin glycoside compounds in liver organization ROS and SOD content impact (compared to normal group,###P<0.001；Compared to model group,*P < 0.05,**P < 0.01,***P<0.001)
Fig. 4 be quercetin glycoside compounds in co-culture model HSC cell number impact (compared to normal group 2,#P<0.05,##P<0.01；Compared to model group,*P<0.05,**P<0.01)
Fig. 5 be quercetin glycoside compounds in co-culture model ROS content impact (compared to normal group 2,###P<0.01；Compared to model group,*P<0.05,***P<0.001)
Fig. 6 be quercetin glycoside compounds in co-culture model TGF-β content impact (compared to normal group 2,##P<0.01；Compared to model group,*P<0.05,**P<0.01) 。
Claims (6)
1. quercetin glycoside is for preparing the purposes of the medicine of preventing/treating hepatic fibrosis disease.
2. the purposes of claim 1, wherein quercetin glycoside has having structure and leads to formula (I):
Wherein R represents-COOH or-CH2X, wherein X represents OH, F, Cl, Br, I, CN, NO2、NH2、CF3、
SH、OCH3、OC2H5Or COOH.
3. the purposes of claim 2, wherein R representative-CH2OH。
4. the purposes of claim 2, wherein R representative-COOH.
5. the purposes of claim 2, wherein R representative-CH2OCH3。
6. the purposes of claim 2, wherein R representative-CH2F。
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102093459A (en) * | 2011-01-10 | 2011-06-15 | 中国人民解放军第二军医大学 | Penthorum chinense pursh extract and preparation method and application thereof |
| CN103951721A (en) * | 2014-05-05 | 2014-07-30 | 南京瑞菁医药科技有限责任公司 | Application of quercetin-O-glucoside derivative to treatment of lipid metabolism disorders |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102093459A (en) * | 2011-01-10 | 2011-06-15 | 中国人民解放军第二军医大学 | Penthorum chinense pursh extract and preparation method and application thereof |
| CN103951721A (en) * | 2014-05-05 | 2014-07-30 | 南京瑞菁医药科技有限责任公司 | Application of quercetin-O-glucoside derivative to treatment of lipid metabolism disorders |
Non-Patent Citations (1)
| Title |
|---|
| 周维晨 等: "老鹰茶总黄酮中山奈酚-葡萄糖苷对TGF-β1诱导的肝星状细胞增殖的影响及机制", 《安徽医科大学学报》 * |
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