CN103638035A - Application of polydatin for preparing medicament for treating atherosclerosis - Google Patents
Application of polydatin for preparing medicament for treating atherosclerosis Download PDFInfo
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Abstract
The invention belongs to a clinic application field of active components of traditional Chinese medicinal materials, especially an application of extract polydatin from the traditional Chinese medicinal material gentrin knotweed for preparing a medicament for treating atherosclerosis. The subject of the invention is the application of extract polydatin from the traditional Chinese medicinal material gentrin knotweed for preparing the medicament for treating atherosclerosis. The polydatin is a product prepared by combining resveratrol with glucose, and belongs to a stilbene compound in the gentrin knotweed components, that is a hydroxy stilbene compound. The medicaments for treating atherosclerosis in the prior art include statins, fibrates, nicotinic acids, unsaturated fatty acid and aspirin, which all have obvious side-effects, such as digestive system reaction, dizziness and rash of statins, and gastric mucosa damage, reduction of platelet and leucocyte, alimentary canal stimulation and asthma induction of aspirin. The polydatin provided by the invention, by action mechanisms of raising a SOD level and recuing a content of oxidation type low density lipoprotein, can effectively inhibit atherosclerosis, and has beneficial effects of high efficiency and safety.
Description
Technical field
The application of the extract polygonin of application, especially the Chinese crude drug Rhizoma Polygoni Cuspidati of the active ingredient that the invention belongs to Chinese crude drug in clinical in preparation treatment atherosclerosis medicine.
Background technology
Rhizoma Polygoni Cuspidati (formal name used at school: Polygonum cuspidatum), originate in East Asia Region, be distributed in the ground such as Jiangsu, Jiangxi, Shandong, Sichuan of area on the south Hokkaido, Japan western part, the Korea peninsula, Taiwan and China.Mildly bitter flavor, cold nature, containing Polydatin, flavone etc.Rhizoma Polygoni Cuspidati has the analgesic therapy of invigorating blood circulation, dampness removing jaundice eliminating, controls the effect of jaundice due to damp-heat, heat-clearing and toxic substances removing, preventing phlegm from forming and stopping coughing, is used for controlling blood stasis amenorrhea, rheumatic arthralgia, traumatic injury, stranguria with turbid discharge leukorrhagia, burn due to hot liquid or fire, sore and toxic, the illness such as venom, cough due to lung-heat.
Summary of the invention
The technical problem that invention solves
The technical problem that invention will solve for to choose a kind of efficient, cheap, safe Chinese medicine in numerous treatment atherosclerosis medicines.
The technical scheme that invention adopts
Choose Chinese crude drug Rhizoma Polygoni Cuspidati, use the polygonin extracting from Rhizoma Polygoni Cuspidati to prepare treatment atherosclerosis medicine.
The beneficial effect of the invention
Existing treatment atherosclerosis medicine has Statins, the special class of shellfish, nicotinic acid class, unsaturated fatty acid and aspirin etc., all has obvious side effect and uses.As the digestive system reaction of Statins, dizzy, erythra, the gastric mucosa injury of aspirin, platelet and leukopenia, digestive tract stimulate and bring out asthma etc.Polygonin of the present invention suppresses atherosclerosis effectively by improving the mechanism of action such as content of the low density lipoprotein, LDL of body SOD level, reduction oxidized form, has the beneficial effect of highly effective and safe.
Accompanying drawing explanation
The situation of change of Mouse Weight after the administration of Fig. 1 polygonin
The changing condition of mice serum middle-high density lipoprotein after the administration of Fig. 2 polygonin
The changing condition of T-CHOL in mice serum after the administration of Fig. 3 polygonin
Oxidized low-density lipoprotein changing condition in mice serum after the administration of Fig. 4 polygonin
The changing condition of total free radical in mice serum after the administration of Fig. 5 polygonin
The changing condition of total antioxidation level in mice serum after the administration of Fig. 6 polygonin
The pathological change situation of mice coronary artery (CA) and ventral aorta (AA) after the administration of Fig. 7 polygonin
The changing condition of Treg cell proportion in mouse peripheral blood after the administration of Fig. 8 polygonin
The changing condition of TH17 cell proportion in mouse peripheral blood after the administration of Fig. 9 polygonin
Specific embodiment embodiment
materials and methods
1.1 laboratory animals:apoE (/-) mice, 32, Mus age 8 week age, entirely male, body weight 18-20g(strain C57BL/6J, purchased from U.S. Jackson laboratory), by Nanjing University's pattern Experimental Animal Center, provided.With 12 of genetic background C57BL/6J mices, Mus age 8 week age, entirely male, body weight 18-20g.Animal production licence number: SCXK (Soviet Union) 2002-0001.
medicine and reagent:polygonin is bought in Chinese pharmaceutical biological product regular inspection institute, cholesterol (Chengdu Ke Long chemical company), fatty acid (commercially available Adeps Sus domestica boils), LTB4 enzyme linked immunological kit is purchased from R & D company, and LTD4 enzyme linked immunological kit is purchased from BD company.RT-PCR test kit is purchased from precious biological engineering (Dalian) company limited (TaKaRa).Western Blot test kit is purchased from green skies biotechnology research institute.Yihong, haematoxylin dyeing is bought and is built up Bioengineering Research Institute from Nanjing.High density lipoprotein detection kit, oxidized low-density lipoprotein test kit, T-CHOL detection kit is bought from U.S. Mercodia company.TH17, Treg flow cytometer detection test kit is bought from U.S. eBioscience company.Total free radical detection kit, SOD test kit is bought from Sheng Xing bio tech ltd, Nanjing.
capital equipmentzEISS microscope, Excelsion dewaterer, Shandon embedding machine, RM2235 microtome, U.S. Thermo company; Electronic balance, German Sartorius company; 2400 type pcr amplification instrument, U.S. Applied Biosystems company;
2 experimental techniques
2.1 high lipid food compound methodshigh lipid food formula is: 1.25% cholesterol, 15% Adeps Sus domestica, 83.75% normal diet.Feed meal is after sufficiently mixing by processing pellet by feed factory.
mice coronary atherosclerosis is set up32 ApoE (/-) mice adaptability is fed after one week and given high lipid food, feed continuously 12 weeks.Choose 12 of the normal C57BL/6J mices in 8 week age of identical genetic background, entirely male, the normal diet of feeding, as Normal group simultaneously.
medication and dosagehigh lipid food ApoE (/-) mice of feeding continuously 12 weeks is dissected to 2 and 2 the normal diet C57BL/6J mices of feeding and get its ventral aorta and do pathology, see whether it has formed atherosclerosis.By forming atherosclerosis ApoE (/-) mice, be divided into model control group, polygonin dosage group, positive drug (simvastatin) matched group, 10 every group.Positive drug (simvastatin) is with the gavage of 0.05g//(kgd), and polygonin is containing crude drug 200mg/ (kgd) gavage, and blank group and model control group give respectively the distilled water of same volume, amounts to 4 weeks.Remove the normal feedstuff feeding that Normal group gives; All the other each groups continue to give high lipid food every day.After 16 weeks, mice fasting after administration the most once be can't help water and is spent the night, and by every group of mice, through 10% chloral hydrate intraperitoneal injection of anesthesia, heart is got whole blood for enzyme-linked immunoassay.Get coronary artery root 0.3cm for pathological analysis, get its ventral aorta and neck arteries and analyze with mRNA.
the pathology detection of coronary atherosclerosisby paraffin embedding after the conventional dehydration of mice coronary artery, from 30 of coronary artery transverse section serial section, every 5, to get 1 and carry out HE dyeing, the meansigma methods that every mice blood vessel is got 6 serial section is analyzed.After application ZEISS microscopic examination is cut into slices and is taken pictures, with the analysis of Image-J image analysis software, calculate meansigma methods analysis, measure plaque area (PA), vessel lumen area (LA), calculation correction plaque area (PA/LA), and represent with percentage ratio (%).
and the ELISA of LTD4 detectsby standing 30 min of mouse blood room temperature, after centrifugal 15 min of 2000 r/min, get serum.By test kit description, detect, application microplate reader is measured absorbance [D (450)] under 450nm wavelength. and using [D (450)] as abscissa, using the concentration value of standard substance as vertical coordinate, thereby draw out standard curve.On coordinate, [D (450)] measure respective concentration value per sample.
lipid determinationmice is put to death, and heart gathers mice whole blood, and a part takes anticoagulant to process, and a part does not take anticoagulant to process.Not anticoagulant part, standing rear centrifugal, 3000 turn, and 10 minutes, extract supernatant, adopt enzyme linked immunosorbent assay to measure: serum total cholesterol, high density lipoprotein, the content of oxidized low-density lipoprotein.Assay method is deferred to the operating procedure of test kit completely, and test kit adopts Mercodia company product.
flow cytometer detectionput to death as mentioned above mice, heart blood sampling, carries out anticoagulant heparin processing, carries out cell counting, according to 2 * 10
6individual cell/200ul system is processed and antibody labeling, according to the step of Treg and TH17 test kit, carries out the detection of Treg and TH17 ratio in blood, and test kit adopts eBioscience company product.Treg adopts CD4+, CD25+, the screening of hiving off of the positive cell of FOXP3+, the screening of hiving off of the positive cell by CD4+ and IL-17+ of TH17 cell.
antioxidant levels detectsput to death as mentioned above mice, heart blood sampling, not anticoagulant, standing rear centrifugal, 3000 turn, and 10 minutes, extract supernatant, adopt enzyme linked immunosorbent assay to measure: total Free Radical Level, SOD antioxidant levels.By test kit description, detect, application microplate reader is measured absorbance [D (450)] under 450nm wavelength. and using [D (450)] as abscissa, using the concentration value of standard substance as vertical coordinate, thereby draw out standard curve.On coordinate, [D (450)] measure respective concentration value per sample.
statistical methoddata with
represent, group difference analysis is carried out in application t check, and application Pearson correlation analysis is carried out the correlation analysis of serum LTB4 and LTD4 level and atheromatous plaque relative area, by SPSS 17.0 statistical softwares, is completed.
result
the situation of change of Mouse Weight after 3.1 polygonin administrations
As can be seen, model group mice average weight presents the situation of rising compared with other group mices, demonstrates after high fat is fed, and can significantly increase the body weight of APOE-/-mice.After polygonin and positive drug treatment, all can reduce the average weight of mice, the low dose group weight loss that wherein demonstrates medicine is particularly evident, and the body weight of other group mices all, in 32g left and right, does not have significant difference statistically.
the changing condition of mice serum middle-high density lipoprotein after 3.2 polygonin administrations
As map analysis; through our statistical analysis; after polygonin Drug therapy; compared with model group, there is approximately 12% rising in the content of the high density lipoprotein in blood; all there is rising in various degree in positive drug group; demonstrate Drug therapy tool and have certain effect (p<0.5), can promote the content of blood middle-high density lipoprotein and then reach the protective effect to blood vessel.
the changing condition of T-CHOL in mice serum after 3.3 polygonin administrations
As map analysis, through our statistical analysis, T-CHOL does not have to occur difference statistically, demonstrates medicine and may not directly reduce the total cholesterol level in blood in each group statistics.
oxidized low-density lipoprotein changing condition in mice serum after polygonin administration
The content of the low density lipoprotein, LDL of oxidized form demonstrates the content of oxidized lipid, and it is the index of lipid peroxidation, is one of index of the incidence of atherosclerosis order of severity.As figure, we can see after Drug therapy, there is the level of lipid peroxidation significantly decline (p<0.5) in polygonin group, demonstrate the anti-oxidation characteristics of polygonin, polygonin is in treatment atherosclerosis, effect declines by a big margin compared with model group, demonstrates polygonin and has good antioxidant effect, prevents and treats the over oxidation of lipid and then reaches atherosclerotic control.
the changing condition of total free radical in mice serum after 3.5 polygonin administrations
As shown in the figure, compared with normal matched group is compared, in the serum of respectively organizing mice of raising through too high fat, the level of free radical is all higher, after polygonin treatment, there is certain decline in the Free Radical Level in blood, wherein polygonin medication therapy groups fall is maximum, and 30% left and right (p<0.5) approximately declines.And positive drug group Free Radical Level does not significantly decrease compared with model group, prompting positive drug can not reduce the Free Radical Level in blood significantly.
the changing condition of total antioxidation level in mice serum after polygonin administration
As shown in the figure, we have detected the SOD level in blood, the analysis found that, and after polygonin treatment, the SOD level in blood significantly raise, and demonstrates polygonin and has good antioxidant effect (p<0.5).And positive drug group is compared with model group, there is no significant difference, prompting positive drug is not brought into play the effect of medicine by antioxidation approach.
the pathological change situation of mice coronary artery (CA) and ventral aorta (AA) after polygonin administration
As shown in the figure, in coronary artery H & E dyeing, can clearly see in the blood vessel of model group and occur a large amount of lipidosiss, and form crystallization, vessel wall thickening, and there is the infiltration of a large amount of inflammatory cells, morbidity is serious.And through after polygonin medication treatment, atheromatous plaque area presents the trend of dwindling, and along with the increase of dosage, the area of atheromatous plaque dwindles gradually.Positive drug prednisone is generally acknowledged fat-reducing medicament, and after drug treatment, although we find to have occurred the crystallization of lipid in blood vessel, blood vessel is still unimpeded, demonstrates prednisone and has certain therapeutical effect.After polygonin drug treatment, atheromatous plaque area significantly dwindles, although speckle disappears not yet completely, but there is not crystallization, demonstrate polygonin and there is good therapeutical effect as anti-oxidation medicine, and after polygonin drug treatment, blood vessel wall is smooth, hypertrophy slows down, and the infiltration of inflammatory cell is lighter.
the changing condition of Treg cell proportion in mouse peripheral blood after polygonin administration
As shown in the figure, through flow cytometry, to the analysis of Treg cell, can find out, polygonin medication therapy groups is compared with model group, there is no significant difference, demonstrates the effect that polygonin does not have significantly to promote Treg cell proportion.
the changing condition of TH17 cell proportion in mouse peripheral blood after polygonin administration
As shown in the figure, through flow cytometry, to the analysis of TH17 cell, can find out, polygonin group, difference (P>0.05) that the equal compared with normal matched group of positive drug treatment group compares that there are no significant, infers that polygonin can not affect the TH17 cellular level of body.
discuss
China is cardiovascular and cerebrovascular disease state occurred frequently.The patient who dies from every year cardiovascular and cerebrovascular disease reaches more than 300 ten thousand people, annual total the more than 50% of death toll of Yi Zhan China.1996---between 2006, China's acute coronary sickness rate is with male average annual 2.3%, women average annual 1.6% speed increase, wherein 45 years old age group acute coronary of male increased by 50%, 35 years old age group acute apoplexy men and women sickness rate has increased respectively 136% and 220%.Atherosclerosis (atherosclerosis, AS) is the common pathogenesis basis of ischemic cerebrocardiac disease.Actively preventing and treating AS, is the important method that reduces the target organ damages such as the heart, brain, kidney and peripheral blood vessel.
The mankind are to existing a century and a half of the research of the disease of this serious harm health of AS, but its definite cause of disease and pathogenesis are not illustrated so far yet completely.The pathogenic hypothesis successively proposing, as " fatty infiltration theory ", " platelet aggregation and thrombosis theory ", " smooth muscle clonal theory ", " vascular endothelial injury reaction theory " etc., these theories connect each other, complement each other.Wherein, damage-reaction hypothesis (response-to-injury hypothesis) that Ross proposed in 1993
[2]by Most scholars, accepted, 1999Ross has further proposed again " atherosclerosis is a kind of chronic inflammatory disease theory " on injury response theory basis.At present, atherosclerosis is the extensive approval that a kind of chronic inflammatory reaction has obtained medical circle, and inflammatory reaction mechanism is the focus of studying at present.Atherosclerotic pathogenesis is quite complicated, thinks that at present inflammatory reaction is one of arteriosclerotic key mechanism of causing a disease.Research thinks, inflammation can promote the abnormal accumulation of aorta lipid, causes atherogenesis.Inflammatory reaction has promoted the formation of atheromatous plaque progress and unstable spot and has broken, has been the major reason that causes cardiocerebrovasculaevents events to occur.
Increasing research in recent years shows, inflammatory and immunoreation play a part very important in the generation of AS and development.Infect the generation of immunity and inflammation promotion AS and develop the control that the proposition of this viewpoint is AS new approaches are provided.Part cardiovascular drugs has been proved and can have regulated inflammatory cell or cytokine network, comprise clinically conventional, angiotensin converting enzyme inhibitor/hypertensinⅡreceptorretarder, B receptor blocking agent, statins etc., therefore, immunologic intervention likely becomes prevention and the diseases related new target drone for the treatment of atherosclerosis.
Intensification along with to atherosclerosis understanding, it is found that immunoreation has dual character, both can promote also can delay the generation of atherosclerosis and complication thereof.Research shows, immunoreation also has study of anti-atherogenic effect simultaneously, and becomes the study hotspot of current immunne response and AS relation.The study of anti-atherogenic effect of CD4+CD25+ regulatory cell (Treg cell).The circulation Tregs that research shows Acute Coronary Syndrome Patients not only quantity reduces but also function reduction.Evidence suggests that Treg cell suppresses atherosclerosis and completes by TGF-β effect and depression effect T cell.Mechanism of action that it is generally acknowledged at present Treg cell has following 3 kinds: 1. externally by cell contact dependent mechanism, play a role.Treg cell can lead to the corresponding receptors bind on CTLA-4, TGF-β and GITR etc. and target cell, and suppresses the expression of IL-2Ra chain on target cell, thereby reduces the propagation of target cell to the reactivity depression effect T cell of IL-2.2. in body, regulatory function mainly relies on the SC factor, as IL-10, IL-4 and TGF-β are subject to the not impact of equal factor of immune response intensity size, regulatory site (as cytokine environment), the SC factor of Treg emiocytosis presents different characteristics in different diseases, and mechanism of action is more complicated.3. Treg cell also can by with antigen presenting cell be used for regulating immunity of organism.
Polygonin is resveratrol (Resveratrol, the product of Res) being combined with glucose, they all belong to the stilbene compound in Rhizoma Polygoni Cuspidati composition, be hydroxy stibene compounds, chemistry by name 3,4 ', 5 one resveratrol one 3 one B-D mono-glucosides (3,4 ', 5 one trihydrox. ystibene mono-3 one p-mono-D-glucoside), structure has JI trans two kinds of Mr390 just.In view of containing 3 phenolic hydroxyl groups in molecule, infer that it may be a kind of oxygen free radical scavenger, realizes its protective effect by antiopxidant effect.At present, polygonin is successively found in belonging to 72 kind of plant in Vitaceae (Parthenocissus, Vitis Amurensis genus, Ampelopsis), Liliaceae (Veratrum, smilax), Polygonaceae (Polygonum, Rheum), pulse family (Sophora, Arachis, Cassia, Trifolium, Bauhinia, Ilex), Deng12 section of ma Yao Jin section (Eucalyptus) 31
This experiment is raised ApoE-/-knock out mice by high fat diet and is set up Atherosclerosis Model, model group mice average weight presents the situation of rising compared with other group mices, demonstrate after high fat is fed, can significantly increase the body weight of APOE-/-mice.After polygonin and positive drug treatment, all can reduce the average weight of mice, the low dose group weight loss that wherein demonstrates medicine is particularly evident, and the body weight of other group mices all, in 32g left and right, does not have significant difference statistically.
After polygonin Drug therapy; compared with model group, there is approximately 12% rising in the content of the high density lipoprotein in blood; all there is rising in various degree in positive drug group; demonstrate Drug therapy tool and have certain effect, can promote the content of blood middle-high density lipoprotein and then reach the protective effect to blood vessel.T-CHOL does not have to occur difference statistically, demonstrates medicine and may not directly reduce the total cholesterol level in blood in each group statistics.
The content of the low density lipoprotein, LDL of oxidized form demonstrates the content of oxidized lipid, and it is the index of lipid peroxidation, is one of index of the incidence of atherosclerosis order of severity.As figure, we can see after Drug therapy, polygonin group occurs that the level of lipid peroxidation significantly declines, demonstrate the anti-oxidation characteristics of polygonin, polygonin is in treatment atherosclerosis, effect declines by a big margin compared with model group, demonstrates polygonin and has good antioxidant effect, prevents and treats the over oxidation of lipid and then reaches atherosclerotic control.
Normal group is compared, in the serum of respectively organizing mice of raising through too high fat, the level of free radical is all higher, and after polygonin treatment, certain decline appears in the Free Radical Level in blood, wherein polygonin medication therapy groups fall is maximum, and 30% left and right approximately declines.And positive drug group Free Radical Level does not significantly decrease compared with model group, prompting positive drug can not reduce the Free Radical Level in blood significantly.
We have detected the SOD level in blood, the analysis found that, and after polygonin treatment, the SOD level in blood significantly raise, and demonstrates polygonin and has good antioxidant effect.And positive drug group is compared with model group, there is no significant difference, prompting positive drug is not brought into play the effect of medicine by antioxidation approach.
In coronary artery H & E dyeing, can clearly see in the blood vessel of model group and occur a large amount of lipidosiss, and form crystallization, vessel wall thickening, and there is the infiltration of a large amount of inflammatory cells, morbidity is serious.And through after polygonin medication treatment, atheromatous plaque area presents the trend of dwindling, and along with the increase of dosage, the area of atheromatous plaque dwindles gradually.Positive drug prednisone is generally acknowledged fat-reducing medicament, and after drug treatment, although we find to have occurred the crystallization of lipid in blood vessel, blood vessel is still unimpeded, demonstrates prednisone and has certain therapeutical effect.After polygonin drug treatment, atheromatous plaque area significantly dwindles, although speckle disappears not yet completely, but there is not crystallization, demonstrate polygonin and there is good therapeutical effect as anti-oxidation medicine, and after polygonin drug treatment, blood vessel wall is smooth, hypertrophy slows down, and the infiltration of inflammatory cell is lighter.
Through flow cytometry, to the analysis of Treg cell, can find out, polygonin medication therapy groups is compared with model group, there is no significant difference, demonstrates the effect that polygonin does not have significantly to promote Treg cell proportion.
To the analysis of TH17 cell, can find out through flow cytometry, polygonin group, difference that the equal compared with normal matched group of positive drug treatment group compares that there are no significant, infers that polygonin can not affect the TH17 cellular level of body.
This experiment adopts polygonin to set up Atherosclerosis Model to raise ApoE-/-knock out mice by high fat diet, detects serum middle-high density lipoprotein, the low density lipoprotein, LDL of oxidized form, the content of T-CHOL; Detect total Free Radical Level in serum, total antioxidation level in serum simultaneously.Detect Treg cell in blood, the change level of TH17 cell, analyzes Treg cell, the effect of TH17 cell in atherosclerosis simultaneously.Adopt H & E dyeing to dye to ventral aorta and coronary artery simultaneously, analyze the size of atherosclerotic plaque and the situation that thickens of blood vessel, analyze polygonin, in atherosclerosis, developing effect occurs.
Claims (1)
1. the application of the extract polygonin of Chinese crude drug Rhizoma Polygoni Cuspidati in preparation treatment atherosclerosis medicine.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113952349A (en) * | 2021-12-01 | 2022-01-21 | 重庆市急救医疗中心 | Application of polydatin in preparation of medicine for treating pulmonary hypertension |
| CN117582450A (en) * | 2024-01-19 | 2024-02-23 | 华医华药(山东)生物医药科技有限公司 | Pharmaceutical composition and preparation for preventing and treating arteriosclerosis, coronary heart disease and gout |
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| CN1742814A (en) * | 2005-09-21 | 2006-03-08 | 蒙一纯 | Knotweed rhizome glycoside dropping pill, capsule, granules and tablet preparing method for treating cardiovascular and cerebrovascular diseases |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113952349A (en) * | 2021-12-01 | 2022-01-21 | 重庆市急救医疗中心 | Application of polydatin in preparation of medicine for treating pulmonary hypertension |
| CN117582450A (en) * | 2024-01-19 | 2024-02-23 | 华医华药(山东)生物医药科技有限公司 | Pharmaceutical composition and preparation for preventing and treating arteriosclerosis, coronary heart disease and gout |
| CN117582450B (en) * | 2024-01-19 | 2024-05-28 | 华医华药(山东)生物医药科技有限公司 | Pharmaceutical composition and preparation for preventing and treating arteriosclerosis, coronary heart disease and gout |
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Application publication date: 20140319 |