CN104434899A - Application of danshinolic acid L in preparing medicament for treating or preventing hepatic fibrosis and renal fibrosis - Google Patents
Application of danshinolic acid L in preparing medicament for treating or preventing hepatic fibrosis and renal fibrosis Download PDFInfo
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Abstract
The invention relates to application of danshinolic acid L or pharmaceutically acceptable salt (comprising alkali metal salts, alkaline-earth metal slats and amine salts), a solvate and a hydrolysable ester in preparing a medicament for treating or preventing hepatic fibrosis and renal fibrosis. The application means that danshinolic acid L has the anti-lipid peroxidation and anti-hepatic injury effects, has the function of alleviating the degree of liver tissues fibrosis and is used for preventing and treating diseases such as hepatic fibrosis, liver cirrhosis and fatty liver, and further has the effect of resisting renal fibrosis.
Description
Technical field
The present invention relates to a kind of novelty teabag of known drug, be specifically related to salvianolic acid L and the application in the medicine preparing prevention or treatment hepatic fibrosis and renal fibrosis of pharmaceutically acceptable salt, solvate and hydrolyzable ester thereof.
Background technology
Hepatic fibrosis is the important pathological characteristics of chronic hepatopathy.The causes of disease such as virus, ethanol, autoimmune disease all can cause hepatic necrosis, regeneration and persistence fibroplasia, finally cause liver cirrhosis.Hepatic fibrosis be chronic hepatopathy to cirrhosis progress must through pathological process, its Fashion and Evolution is the key affecting hepatopathy prognosis, lapse to.Now confirmed that hepatic fibrosis is reversible pathological changes, liver cirrhosis is then irreversible.Therefore, in the therapeutic process of chronic hepatopathy, the prevention of hepatic fibrosis occupies critical role.
Renal fibrosis (comprising kidney region fibrosis and glomerular sclerosis) is the main pathological basis of the kidney damage final stage that a variety of causes causes, renal fibrosis mechanism is comparatively complicated, relevant with many factors, the wherein main and celliferous increment of extracellular matrix-cell and activation, the conversion of vaso-active substance, cytokine and extracellular matrix is unbalance relevant, and kidney region fibrosis is almost the common pathway that all former or secondary kidney disease proceed to end stage renal failure.
Salvianolic acid L (magnesium tanshinoate L) is the new salvianolic acid compound that extraction and isolation goes out in the dry root and rhizome of Chinese medicine labiate Radix Salviae Miltiorrhizae (Salviamiltionrrhia Bge.), open in Chinese patent 2010101358006.Its structural formula following (I),
Its pharmaceutically acceptable salt, solvate and hydrolyzable ester is also comprised in this patent.And demonstrate salvianolic acid L there is antioxidation, effect of scavenging radical.
The present invention finds unexpectedly, and salvianolic acid L has the purposes of prevention and therapy hepatic fibrosis and renal fibrosis.
Summary of the invention
The invention provides a kind of salvianolic acid L or the application in preparation treatment or the Fibrotic medicine of prevention organ of its pharmaceutically acceptable salt, solvate and hydrolyzable ester.
Application of the present invention, described organ fibrosis is hepatic fibrosis or renal fibrosis.
Application of the present invention, described application is the increment that salvianolic acid L can suppress hepatic stellate cell.
Application of the present invention, described application is that salvianolic acid L can reduce Serum ALT, AST enzymatic activity.
Application of the present invention, described application is the content that salvianolic acid L can suppress liver Hyp.
Application of the present invention, described application is that salvianolic acid L can alleviate extent of liver fibrosis.
Application of the present invention, described application is that salvianolic acid L can suppress the deposition of III Collagen Type VI in liver.
Application of the present invention, described application is that salvianolic acid L can anti peroxidation of lipid.
Application of the present invention, described application is that salvianolic acid L has anti-liver cirrhosis, the effect of fatty liver and hepatic injury.
Application of the present invention, described medicine is using salvianolic acid L or its pharmaceutically acceptable salt, solvate and hydrolyzable ester as sole active agent or the pharmaceutical composition as one of active component.
Medicine of the present invention comprises the form of salvianolic acid L or its pharmaceutically acceptable salt, solvate and hydrolyzable ester.Wherein said salvianolic acid L pharmaceutically acceptable salt comprises the conventional salt generated from pharmaceutically acceptable inorganic or organic base, is obtained by the salifying method preparation of routine.The example of salt be applicable to comprises the salt of sodium, potassium, lithium, magnesium, aluminum, calcium, zinc etc., or and N, N '-dibenzyl-ethylenediamin, chloroprocaine, choline, diethanolamine, ethylenediamine, N-METHYL-ALPHA-L-GLUCOSAMINE, procaine, berberine salt.The solvate of described salvianolic acid L is prepared by the method for routine, (solvate be applicable to comprises hydrate, alcohol adduct, or the solvate formed with acetone, oxolane, dichloromethane, dimethyl sulfoxide).
Salvianolic acid L of the present invention is suitable for the form administration with pharmaceutical composition.This based composition can physiologically acceptable carrier or mixed with excipients use with one or more in a usual manner.If likely treatment on using salvianolic acid L of the present invention as crude drug administration, preferred active component is directly as pharmaceutical preparation.In compatible with other compositions and harmless to its pill taker meaning, carrier must be pharmaceutically acceptable.
Pharmaceutical composition of the present invention, can be prepared into pharmaceutical dosage forms in use as required, as oral dosage form, and injection form, vagina or supp anal form etc., optimizing injection form.
Pharmaceutical composition of the present invention, when being prepared into pharmaceutical preparation, can adding as required and will learn acceptable carrier.
Pharmaceutical composition of the present invention, medicine acceptable carrier can be contained as required, wherein salvianolic acid L or its pharmaceutically acceptable salt, solvate and hydrolyzable ester are as active constituents of medicine, its in the formulation shared percentage by weight can be 0.1-99.9%, all the other are medicine acceptable carrier.Pharmaceutical preparation of the present invention, exists in a unit, and described unit dosage form refers to the unit of preparation, as every sheet of tablet, and every capsules of capsule, every bottle of oral liquid, granule every bag, often propping up of injection.
Pharmaceutical composition of the present invention can be any pharmaceutically useful dosage form, and these dosage forms comprise: tablet, sugar coated tablet, film coated tablet, enteric coated tablet, capsule, hard capsule, soft capsule, oral liquid, suck agent, granule, electuary, pill, powder, unguentum, sublimed preparation, suspensoid, powder, solution, injection, suppository, ointment, plaster, cream, spray, drop, drop pill, patch.
Pharmaceutical composition of the present invention, the preparation of its oral administration can containing conventional excipient, and such as binding agent, filler, diluent, tablet agent, lubricant, disintegrating agent, coloring agent, flavoring agent and wetting agent, can carry out coating to tablet if desired.
The filler be suitable for comprises cellulose, mannitol, lactose and other similar filler.Suitable disintegrating agent comprises starch, polyvinylpyrrolidone and starch derivatives, such as sodium starch glycollate.Suitable lubricant comprises, such as magnesium stearate.The suitable acceptable wetting agent of medicine comprises sodium lauryl sulphate.By mixing, fill, the method that tabletting etc. are conventional prepares solid oral composition.Repeatedly mix and active substance can be made to be distributed in those compositionss of a large amount of filler of whole use.
The form of oral liquid can be such as aqueous or oily suspensions, solution, Emulsion, syrup or elixir, or can be the composite dry products of a kind of available water before use or other suitable carrier.This liquid preparation can containing conventional additive, such as suspending agent, such as sorbitol, syrup, methylcellulose, gelatin, hydroxyethyl-cellulose, carboxymethyl cellulose, aluminium stearate gel or hydrogenated edible fats, emulsifying agent, such as lecithin, anhydro sorbitol monooleate or arabic gum; Non-aqueous carrier (they can comprise edible oil), the oily ester of the such as ester of almond oil, fractionated coconut oil, such as glycerol, propylene glycol or ethanol; Antiseptic, such as para hydroxybenzene methyl ester or propyl p-hydroxybenzoate or sorbic acid, and if need, can containing conventional flavouring agent or coloring agent.
For injection, the fluid unit dosage form of preparation contains active substance of the present invention and sterile carrier.According to carrier and concentration, this compound can be suspended or dissolve.The preparation of solution is normally by being dissolved in a kind of carrier by active substance, filter-sterilized before being loaded a kind of suitable bottle or ampoule, then seals.Adjuvant such as a kind of local anesthetic, antiseptic and buffer agent also can be dissolved in this carrier.In order to improve its stability, by freezing for this compositions after loading bottle, and under vacuo water can be removed.
Pharmaceutical composition of the present invention, applicable medicine acceptable carrier is optionally added when being prepared into medicament, described medicine acceptable carrier is selected from: mannitol, sorbitol, sodium pyrosulfite, sodium sulfite, sodium thiosulfate, cysteine hydrochloride, TGA, methionine, vitamin C, EDETATE SODIUM, Ethylenediaminetetraacetic Acid Calcium Salt, the alkali-metal carbonate of monovalence, acetate, phosphate or its aqueous solution, hydrochloric acid, acetic acid, sulphuric acid, phosphoric acid, aminoacid, sodium chloride, potassium chloride, sodium lactate, xylitol, maltose, glucose, fructose, dextran, glycine, starch, sucrose, lactose, mannitol, silicon derivative, cellulose and its derivates, alginate, gelatin, polyvinylpyrrolidone, glycerol, POLYSORBATE 80, agar, calcium carbonate, calcium bicarbonate, surfactant, Polyethylene Glycol, cyclodextrin, beta-schardinger dextrin-, phospholipid material, Kaolin, Pulvis Talci, calcium stearate, magnesium stearate etc.
Pharmaceutical preparation of the present invention according to the situation determination usage and dosage of patient, can take three every day in use, each 1-20 agent, as: 1-20 bag or grain or sheet, and every agent 1mg-1000mg.
Medicine provided by the invention when for prevention and therapy hepatic fibrosis and renal fibrosis, for the dosage of adult treatment usually by the scope in 0.02-5000mg every day, preferred 1-1500mg every day.Required dosage can be single dosage or metering repeatedly, by suitable doses at intervals, such as twice daily, three times, four times or more.The active component of 0.1-99% can be contained according to preparation of the present invention, to the preferred 30-95% of Tablet and Capsula agent, the preferred 3-50% of liquid preparation.
Pharmaceutical applications of the present invention is proved by following experiment:
Experimental example 1: salvianolic acid L Against Hepatic Fibrosis in Vitro effect
Salvianolic acid L carries out cytoactive Inhibition test to the cultured rat hepatic stellate cells of In vitro culture and hepatic parenchymal cells respectively.
1.1 Experimental agents and reagent
Salvianolic acid L provided by Tianjin Tian Shili group academy modern Chinese medicine, content more than 95%; DMEM, RPMI1640, Hank ' s liquid, hyclone (FBS), antibiotic be purchased from Gibco company, and MTT tetramethyl tetrazolium bromide is purchased from Sigma company.Analytical pure DMSO is purchased from Tianjin analytical reagent one factory.
1.2 cell strain
Cultured rat hepatic stellate cells strain (HSC-T6) and people's hepatic parenchymal cells strain (L02) are all purchased from institute of oncology of Chinese tumour hospital.
1.3 experimental technique
Cultured rat hepatic stellate cells strain (HSC-T6) and people's hepatic parenchymal cells strain (L02) all adopt RPMI1640 complete medium to cultivate.All cells is cultivated all at 37 DEG C of 5%CO
2cultivate under condition, after cell attachment reaches 80% fusion, culture medium of inclining, Hank ' s liquid cleaning twice, use 0.25% trypsinization, be mixed with certain density cell suspension, be inoculated in 96 hole ELISA Plate, every hole adds 100 μ L, and cell seeding density is 1 × 10
3cells/ hole, in 37 DEG C of 5%CO
2cultivate 24 hours.
Change the fresh medium 200 μ L containing variable concentrations given the test agent (DMSO final concentration≤0.1%) and DMSO contrast after 24h, establish 4 dosage groups by reagent, each concentration establishes 3 parallel holes, and establishes blank well to return to zero.In 37 DEG C of 5%CO
2after cultivating 72hr, every hole adds serum-free medium freshly prepared 5mg/mL MTT 10 μ L, continue to cultivate 4h, every hole adds 100 μ LDMSO and dissolves MTT formazan granule, after microoscillator vibration mixing, under determined wavelength 578nm, measure OD value by microplate reader, calculate given the test agent to cell inhibitory rate and half-inhibition concentration (IC
50).
1.4 experimental result
The main cellulation of the extracellular matrix such as collagen when hepatic stellate cell is hepatic fibrosis.Salvianolic acid L can suppress the increment of hepatic stellate cell, IC
50be 2.43 μ g/ml, to hepatic parenchymal cells growth unrestraint effect.
Table 1 salvianolic acid L is to the inhibitory rate of cell growth of cultured rat hepatic stellate cells
Experimental example 2: salvianolic acid L is to the protective effect of acute liver injury of rats
2.1 Experimental agents and reagent
Salvianolic acid L provided by Tianjin Tian Shili group academy modern Chinese medicine, content more than 95%; Carbon tetrachloride (CCl
4): East China, Tianjin chemical reagent work, import subpackage, analytical pure.
2.2 laboratory animal
SPF level male SD rat 150, body weight 180 ~ 220g.Be purchased from Beijing company of dimension tonneau China, the animal quality certification number: SCXK (capital) 2007-0001.Rearing conditions: raise in air-conditioned animal housing, temperature is 20 DEG C ~ 25 DEG C, relative humidity 60%, 5, every cage, lighting hours 12 hours, adds feedstuff at regular time and quantity, edible Mus special feed (production of Beijing Ke Aoxie feed corporation,Ltd), freely drink water, every day changes bedding and padding.
2.3 experimental technique
2.3.1 grouping is tested
Weighed by animal, numbering, by body weight random packet.Whole experiment is divided into group, is respectively normal group, model group, salvianolic acid L high dose group (5mg/kg), salvianolic acid L low dose group (2.5mg/kg), often organizes 10.All adopt tail vein injection administration.
2.3.2 model preparation
In administration fasting in the 4th day 24 hours, the 5th day 1h employing 25%CCl after last administration
4peanut oil solution is according to 0.5ml/100g subcutaneous injection.After modeling, 18 hours ophthalmic corners of the eyes are got blood and are about 1ml, and the centrifugal 10min of 3500r/min, gets serum-20 DEG C frozen.
2.3.3 Testing index
Full automatic biochemical apparatus is adopted to measure the content of serum alt, AST.
2.3.4 data statistical approach
When initial data arranges, with 95% confidence interval definition abnormal data, exceed by numerical value
extraneous data are rejected.SPSS11.5 software is adopted to carry out statistical analysis.Measurement data with
represent, between model group and Normal group, adopt independent samples t test, whether successful to observe modeling.Subsequently with each medicine group compared with model group, adopt one factor analysis of variance, to evaluate the effect of medicine.P<0.05 is that difference has significant.
2.4 experimental result
Confirm through the research of carbon tetrachloride Acute Hepatic loss rat experiment, salvianolic acid L significantly can reduce Serum ALT, AST enzymatic activity, has the effect of significant anti-liver injury.
Table 2 salvianolic acid L/M is to CCl
4the protective effect of the acute liver injury of rats caused (
)
Compare with model group,
*p<0.01
Experimental example 3: salvianolic acid L is on the impact of rat liver fibrosis
3.1 Experimental agents and reagent
Salvianolic acid L provided by Tianjin Tian Shili group academy modern Chinese medicine, content more than 95%; Colchicine (Colchicine, Col) is Sigma product; N-nitrosodimethylamine (DMN) is purchased from Chengdu Shuo Long trade Co., Ltd, analytical pure AR content 99.0 (%), CAS 62-75-9; Pc III (III Collagen Type VI) detection kit, is purchased from Hermes Criterion Biotechnology, lot number: 742RA1026489; Hyp (hydroxyproline) detection kit builds up Bioengineering Research Institute purchased from Nanjing, lot number 20120620.
3.2 laboratory animal
SPF level male SD rat 150, body weight 180 ~ 220g.Be purchased from Beijing company of dimension tonneau China, the animal quality certification number: SCXK (capital) 2007-0001.Rearing conditions: raise in air-conditioned animal housing, temperature is 20 DEG C ~ 25 DEG C, relative humidity 60%, 5, every cage, lighting hours 12 hours, adds feedstuff at regular time and quantity, edible Mus special feed (production of Beijing Ke Aoxie feed corporation,Ltd), freely drink water, every day changes bedding and padding.
3.3 experimental technique
3.3.1 grouping is tested
Weighed by animal, numbering, by body weight random packet.Whole experiment is divided into group, is respectively normal group, model group, positive controls (colchicine), salvianolic acid L high dose group (10mg/kg), salvianolic acid L low dose group (5mg/kg), often organizes 10.All adopt tail vein injection administration.
3.3.2 model preparation
With 0.5%DMN lumbar injection, 0.2ml/100g; 1 time/d, continuous 3d, totally 3 weeks, prepares Rat Liver Fibrosis Model weekly.In modeling random packet, give respective medicine respectively, 1 time/d, totally 3 weeks.After last administration, 24 hours postcava get blood, and the centrifugal 10min of 3500r/min, gets serum-20 DEG C frozen.Get hepatic tissue, part is carried out formalin and is fixed, and does paraffin section, and conventional H E dyes, and tissues observed pathology form under optical microscope, fibroplasia degree is divided into 0-4 level.Another part prepares 10% tissue homogenate, and-20 DEG C frozen.
3.3.3 Testing index
Adopt full automatic biochemical apparatus to measure serum alt, AST content, the content of Procollagen III peptid or its metabolism fragment (P III NP) and organize Hyp content, all measure by detection kit assay method.
3.3.4 data statistical approach
When initial data arranges, with 95% confidence interval definition abnormal data, exceed by numerical value
extraneous data are rejected.SPSS11.5 software is adopted to carry out statistical analysis.Measurement data with
represent, between model group and Normal group, adopt independent samples t test, whether successful to observe modeling.Subsequently with each medicine group compared with model group, adopt one factor analysis of variance, to evaluate the effect of medicine.P<0.05 is that difference has significant.
3.4 experimental result
Confirm through N-nitrosodimethylamine loss Rat Liver Fibrosis Model experimentation, salvianolic acid L significantly can suppress the content of rat liver Hyp, alleviates extent of liver fibrosis, suppresses the deposition of III Collagen Type VI in liver, there is the effect of significant anti-hepatic fibrosis, and have certain dose-effect relationship.Significantly reduce Serum ALT, AST enzymatic activity simultaneously, alleviate the degree of hepatic tissue steatosis, have the effect of significant anti peroxidation of lipid, anti-liver injury.
Pathological examination display rats in normal control group liver structure is normal; All there is obvious fibrosis in model group rats liver; In each group of salvianolic acid L, all comparatively model group is light for fibrosis.
Table 3 salvianolic acid L is on the impact of hepatic fibrosis rats liver Serum ALT, AST and P III NP content
Compare with model group,
*p<0.05,
*p<0.01
Table 4 salvianolic acid L is on the impact of liver tissues of rats with hepatic fibrosis HYP content
Compare with model group,
*p<0.05,
*p<0.01
Table 5 salvianolic acid L is on the impact of hepatic fibrosis rats Serum ALT, AST, Pc III
Experimental example 4: salvianolic acid L is to the fibroblastic cell proliferation experiment of Ren Mus.
4.1 Experimental agents and reagent
Salvianolic acid L provided by Tianjin Tian Shili group academy modern Chinese medicine, content more than 95%.DMEM, RPMI1640, Hank ' s liquid, hyclone (FBS), antibiotic be purchased from Gibco company.MTT tetramethyl tetrazolium bromide is purchased from Sigma company.Analytical pure DMSO is purchased from Tianjin analytical reagent one factory.Fn Fiberonectin (Fn) ELISA kit is purchased from Shanghai sun biotech company.
4.2 cell
Ren Mus fibroblast is all purchased from institute of oncology of Chinese tumour hospital.
4.3 experimental technique
Ren Mus fibroblast is inoculated in 75ml culture bottle that (density is every bottle 2 × 10
6), cultivate in 37 DEG C of 5%CO2 incubators.Become after monolayer until cell confluency, digest 3min with 0.25% trypsin be dissolved in without in Hank ' the s balanced salt solution of calcium magnesium in room temperature, be prepared into cell suspension, go down to posterity when reaching 70% ~ 80% density.
Getting the cell being in exponential phase is inoculated in 96 orifice plates, and every hole adds cell suspension 100ml, and density is every ml 1 × 10
5individual, in 37 DEG C, after cultivating 24 h in 5%CO2 incubator, abandon supernatant.Add the salvianolic acid L of variable concentrations respectively, matched group only adds the RPMI1640 culture medium (every hole 100ul) of 10% calf serum, and each concentration establishes 5 multiple holes.In 37 DEG C, cultivate in the CO2 incubator of 5%, cultivate 20h respectively, add MTT20ul (1mg/ml) after 44h, then put in incubator and take out culture plate after 4h, after abandoning supernatant, every hole adds DMSO 100ul, measure each hole optical density value (A value) by microplate reader, namely A 570-A 590nm reflects the propagation degree of cell.
ELISA method is adopted to measure the expression of Fn.Get the every ml 1 × 10 of Ren Mus fibroblast cell of the salvianolic acid L adding variable concentrations respectively
5individually be inoculated in 96 orifice plates, put into 37 DEG C, cultivate 48h again in 5%CO2 incubator, get cell conditioned medium liquid and add in instrument connection, the method provided according to Fn detection kit operates.With standard curve control after microplate reader mensuration optical density value, draw the numerical value of Fn.
4.4 data statistical approach
When initial data arranges, with 95% confidence interval definition abnormal data, exceed by numerical value
extraneous data are rejected.SPSS11.5 software is adopted to carry out statistical analysis.Measurement data with
represent, between model group and Normal group, adopt independent samples t test, whether successful to observe modeling.Subsequently with each medicine group compared with model group, adopt one factor analysis of variance, to evaluate the effect of medicine.P<0.05 is that difference has significant.
4.5 experimental result
Salvianolic acid L is on the impact of kidney of rats fibroblast proliferation: after salvianolic acid L and solvated compounds various dose group act on Ren Mus fibroblast 48h, all obviously can suppress the fibroblastic propagation of Ren Mus, difference significance (P<0.01) compared with matched group.Refer to table 6.
Table 6 salvianolic acid L to the Fibrotic protective effect of kidney of rats (
)
Compare with model group,
*p<0.01,
*p<0.05.
4.6 discuss
Kidney region fibrosis is the final result of the renal tubules that causes of a variety of causes and interstitial pathological changes, is also one of major reason causing End-stage renal failure.Kidney region fibrosis is the co-channel that various kidney disease develops into End-stage renal failure, and its principal character is a large amount of depositions of a large amount of propagation of Stromal fibroblasts and the extracellular matrix (ECM) of fibroblasts to secrete.Current research shows, when various stimulating factor causes kidney fibroblast proliferation, can secrete a large amount of Fn, cause ECM produce increase, minimizing of degrade, formation kidney region fibrosis.Therefore suppress the fibroblastic propagation of kidney, reduce the secretion of Fn, even reverse in kidney region fibrosis process in prevention and play vital effect.
This research shows, after salvianolic acid L and solvated compounds various dose group effect kidney fibroblast 48h, obviously can suppress the fibroblastic propagation of kidney, reduces the secretion of Fn, demonstrates the effect suppressing kidney fibroblast proliferation.
Experimental example 5: anti-renal fibrosis effect in salvianolic acid L body
5.1 tested material
Salvianolic acid L provided by Tianjin Tian Shili group academy modern Chinese medicine, content more than 95%.Fosinopril, Shanghai Shi Guibao pharmaceutical Co. Ltd of Sino-U.S. provides (lot number is 1001091)
5.2 laboratory animal
SPF level male SD rat 70, body weight 180 ~ 220g.Be purchased from Beijing company of dimension tonneau China, the animal quality certification number: SCXK (capital) 2007-0001.Rearing conditions: raise in air-conditioned animal housing, temperature is 20 DEG C ~ 25 DEG C, relative humidity 60%, 5, every cage, lighting hours 12 hours, adds feedstuff at regular time and quantity, edible Mus special feed (production of Beijing Ke Aoxie feed corporation,Ltd), freely drink water, every day changes bedding and padding.
5.3 reagent
SP test kit, DAB colour reagent box, haematoxylin, Foochow steps new company and provides.Rabbit Chinese People's Anti-Japanese Military and Political College Mus α-SMA polyclonal antibody, Bo Aosen bio tech ltd, Beijing provides.Rabbit Chinese People's Anti-Japanese Military and Political College Mus E-cadherin polyclonal antibody, anti-, the antibody diluent of horseradish peroxidase-labeled goat-anti rabbit two, Wuhan doctor's moral company provides.
5.4 instrument
Sheet machine baked by ASP300 fully-automatic dewatering machine, automatically embedding machine, constant temperature push jack, sliding microtome is German Lycra company, table-type high-speed refrigerated centrifuge (5810 type), Eppendorf, Germany; Nikon 80i Pathologic image analysis system, Japanese Nikon company.
5.5 experimental technique
With chloral hydrate (350mg/kg) intraperitoneal anesthesia experimental rat; get rats with left Fu Shen district otch; left side ureter is separated with ophthalmic tweezers; with No. five surgical threads; at renal calices place and inferior pole of kidney place ligation ureter respectively, but not from disconnected ureter, note during operation not damaging kidney peplos and protecting surrounding tissue; Post operation is placed in original position kidney, layer-by-layer suture peritoneum, muscle, skin.Sterile working is strictly observed in art.
After modeling, animal is weighed, numbering, 7 groups are divided at random by body weight, be respectively Normal group, model control group, positive drug control group (6.6mg/kg), salvianolic acid L low dose group (2.5mg/kg), salvianolic acid L high dose group (5.0mg/kg), solvated compounds low dose group (2.5mg/kg), solvated compounds high dose group (5.0mg/kg), often organize 10.All adopt tail vein injection administration.
Tail vein injection administration is after the 10th day, under narcotism, cut animal abdominal cavity open, peel off whole intestinal tissue, get ligation side kidney after ventral aorta blood sampling, be placed in and make 4 μm of paraffin sections after 4% paraformaldehyde fixedly carries out ethanol dehydration, paraffin embedding and carry out immuning tissue's dyeing.
Immunohistochemical detection nephridial tissue α-SMA expresses.α-SMA is 1:50 dilution, operates in strict accordance with SABC process.SABC positive findings is that Cytoplasm and renal interstitial dye brown color or yellow particle, HPIAS-1000 computer picture automatic analysis system is adopted to analyze each group of image, often open section under 400 times of visuals field, get 5 visuals field at random, calculate the average gray level in each visual field, be divided into 0-255 level totally 256 grades, average gray value is higher, shows that its light transmittance is stronger, immunohistochemical staining is more shallow, and positive rate is lower.
5.6 data statistical approach
When initial data arranges, with 95% confidence interval definition abnormal data, exceed by numerical value
extraneous data are rejected.SPSS11.5 software is adopted to carry out statistical analysis.Measurement data with
represent, between model group and Normal group, adopt independent samples t test, whether successful to observe modeling.Subsequently with each medicine group compared with model group, adopt one factor analysis of variance, to evaluate the effect of medicine.P<0.05 is that difference has significant.
5.7 experimental result
E-cadherin semi-quantitative analysis result: compare with normal group, all the other are the rising of group nephridial tissue E-cadherin gray value level respectively, difference has statistical significance (P < 0.05), illustrates that modeling renal tissues of rats Ecadherin positive expression reduces.Compare with model group, each treatment group Ecadherin expresses gray value level to be had and reduces in various degree, difference has statistical significance (P < 0.05), illustrates that each treatment group renal tissues of rats E-cadherin positive expression strengthens in various degree.Refer to table 2.
α-SMA semi-quantitative analysis result: compare with normal group, model group nephridial tissue α-SMA gray value level reduces, and difference has statistical significance (P < 0.05), and modeling success is described.Compare with model group, each treatment group α-SMA expresses gray value level to be had and raises in various degree, and difference has statistical significance (P < 0.05), illustrates that each treatment group renal tissues of rats α-SMA positive expression reduces in various degree.Illustrate that fosinopril group and each administration group have and suppress nephridial tissue α-SMA expressional function, and effect is suitable.Refer to table 7.
Table 7 salvianolic acid L to renal tissues of rats α-SMA, E-cadherin express gray value level compare (
)
Note: compare with normal group, * P < 0.05; Compare with model group, #P < 0.05.
5.8 discuss
Along with doctor trained in Western medicine prevents and treats chronic kidney disease progress day by day deeply, the molecular mechanism EMT finding to cause renal fibrosis to occur main with TGF-β/Smad, Jak/Stat, Wnt/ β-catenin etc. many signal paths relevant, suppress EMT to occur, become the key for the treatment of chronic nephropathy.Main process occurs EMT is that renal cells E-cadherin expresses decline, and transform to fibroblast, fibroblast obtains some phenotype of smooth muscle cell, and become myofibroblast, its mark α-SMA expresses increase.Myofibroblast produces and secretes a large amount of cytoplasmic matrix, the excessive accumulation of cytoplasmic matrix and cause renal fibrosis to increase the weight of.
Salvianolic acid L administration group compares with model group, each treatment group Ecadherin expresses gray value level to be had and reduces in various degree, difference has statistical significance (P < 0.05), illustrates that each treatment group renal tissues of rats E-cadherin positive expression strengthens in various degree.α-SMA semi-quantitative analysis found that simultaneously, compares with normal group, and model group nephridial tissue α-SMA gray value level reduces, and difference has statistical significance (P < 0.05), and modeling success is described.Compare with model group, each treatment group α-SMA expresses gray value level to be had and raises in various degree, and difference has statistical significance (P < 0.05), illustrates that each treatment group renal tissues of rats α-SMA positive expression reduces in various degree.Illustrate that fosinopril group and each administration group have and suppress nephridial tissue α-SMA expressional function, and effect is suitable.In sum, salvianolic acid L can alleviate the generation of renal fibrosis by suppressing the generating process of EMT, and has good curative effect.
The Fibrotic antagonism research of kidney of rats that experimental example six salvianolic acid L induces Aristolochic Acid
6.1 tested material
Salvianolic acid L provided by Tianjin Tian Shili group academy modern Chinese medicine, content more than 95%.Fosinopril, Shanghai Shi Guibao pharmaceutical Co. Ltd of Sino-U.S. provides (lot number is 1001091)
6.2 laboratory animal
SPF level male SD rat 70, body weight 180 ~ 220g.Be purchased from Beijing company of dimension tonneau China, the animal quality certification number: SCXK (capital) 2007-0001.Rearing conditions: raise in air-conditioned animal housing, temperature is 20 DEG C ~ 25 DEG C, relative humidity 60%, 5, every cage, lighting hours 12 hours, adds feedstuff at regular time and quantity, edible Mus special feed (production of Beijing Ke Aoxie feed corporation,Ltd), freely drink water, every day changes bedding and padding.
6.3 reagent:
Aristolochic Acid (AA): Sigma-Aldrich Products, containing 29%AA I and 61%AA II; Serum creatinine (Scr) test kit, blood urea nitrogen (BUN) test kit are purchased from Nanjing and build up.
6.4 experimental technique
Laboratory animal, through the laundering period of 1 week, after carrying out body weight equilibrium, is divided into 7 groups at random.Be respectively Normal group, model control group, positive drug control group (6.6mg/kg), salvianolic acid L low dose group (2.5mg/kg), salvianolic acid L high dose group (5.0mg/kg), solvated compounds low dose group (2.5mg/kg), solvated compounds high dose group (5.0mg/kg), often organize 10.Model group accepts AA gavage every day, and dosage is 5mgkg-1, and gavage volume is 1ml100g-1.AA becomes suspension (lower same) for subsequent use with normal saline before gavage.After each treatment group accepts AA gavage (5mgkg-1) every day, tail vein injection is medicine separately.Normal group (n=8): every day tail vein injection equivalent normal saline.Each group of process all continues 8 weeks, weighs weekly the weight of animals to adjust dosage.Draw materials after last administration, Immunohistochemical Method detects renal tubular interstitium III Collagen Type VI (Col-III), fibronectin (FN), α-smooth muscle actin (α-SMA) expression, adopts Electronic Speculum to carry out histology to kidney.
6.5 statistical methods adopt SPSS 14.0 statistical software, and data represent with x ± s.Comparison between each group adopts one factor analysis of variance (ANOVA) inspection.
6.6 experimental result
6.6.1 renal tissue FN, III Collagen Type VI and α-SMA express
Adopt semi-quantitative analysis, often organize random selecting 9 visuals field, calculate positive area and express.Result normal group renal interstitial only has FN and Col-III expression of collagen of minute quantity, model group significantly increases with normal group (P<0.01, P<0.001), salvianolic acid L high dose group significantly reduces (P<0.05, P<0.01) with model group.Normal rats renal interstitial only has a small amount of α-SMA to express, model group significantly increases with normal group (P<0.01), and salvianolic acid L low dose group and high dose group significantly reduce (P<0.05 with model group; P<0.01) (table 8).
The impact that table 8 salvianolic acid L organizes FN, III Collagen Type VI and α-SMA to express on renal fibrosis rat kidney
Compare with model group: *: P<0.05; *: P<0.01; * *: P<0.001
6.6.2 renal tissue electron microscopic examination
Normal group glomerule normal in size, nucleus is complete, and nuclear membrane is clear; Epithelial cell without hypertrophy, without swelling; Basement membrane is smooth, without swelling, deposits without electron-dense thing; Renal tubules tube chamber is without expansion, and epithelial cell is without swelling; Interstitial is without leukocyte infiltration, edema, fibrosis, and interstitial mitochondrial size and form is normal, microvillus queueing discipline; Renal tubular basement membrane deposits without electron-dense thing.Model group glomerule increases, and the swelling of basement membrane severe, inside has electron-dense thing to deposit; The swelling of renal cells severe, the distortion of interstitial mitochondrial, swelling, the swelling of part microvillus, arrangement disorder, has electron-dense thing to deposit in some renal tubular basement membrane; In renal interstitial, the fibroblast of visible hyperplasia and mononuclear phagocyte infiltrate, the collagen fiber of regular arrangement in endochylema.Salvianolic acid L high and low dose group and solvated compounds high and low dose group comparatively model group have clear improvement, the swelling of part glomerular basement membrane, have electron-dense produce raw in some basement membrane; Renal cells swelling is out of shape, and mitochondrion distortion, swelling, microvillus arrangement disorder, has the fat granule of distortion to deposit in some renal tubules.
7. discuss
According to the literature, Stem of Aristolochia manshuriensis can cause nephrotoxicity, and wherein Aristolochic Acid (AA) is its main component.Low dose of or normal dose long-term therapy takes the Chinese medicine containing AA composition, no matter pill or decoct, and single or compound recipe, all can accumulate in vivo and cause chronic poisoning, being developed to chronic renal failure.This experiment adopts the mode of AA gavage modeling, prepares renal fibrosis model.This model turns to main manifestations with acute and chronic renal tubulointerstitial lesion and renal interstitial Progressive symmetric erythrokeratodermia fiber on pathology, similar with the characteristics of incidence of acute and chronic renal failure and renal tubular function obstacle clinically.
After salvianolic acid L and solvated compounds administration, obviously can improve nephridial tissue FN, III Collagen Type VI and α-SMA in the renal fibrosis model caused due to AA and express (P<0.05; P<0.01), and improve the situation of the swelling of renal tissue basement membrane and electron-dense thing deposition, there is the effect of certain anti-renal fibrosis.
Detailed description of the invention
Prepared by embodiment 1 salvianolic acid L freeze-dried powder
The preparation of salvianolic acid L freeze-dried powder
Prescription:
Technique:
Take adjuvant mannitol in salvianolic acid L and prescription, inject and use water 1500ml, stirring and dissolving, 0.5 gram of activated carbon stirs decolouring 20 minutes, and 0.45 micrometer Millipore filter membrane decarburization, moisturizing is to 2000 milliliters, and aseptic filtration, subpackage, lyophilization, to obtain final product.
The preparation of embodiment 2 salvianolic acid L injection
Prescription:
Get salvianolic acid L, inject and make dissolving in right amount with water 1000ml, stir evenly; Separately get mannitol, inject and make dissolving with water 500ml, add in above-mentioned solution, stir evenly, 0.5 gram of active carbon insulated and stirred 20 minutes, filter, filtrate adjust ph is 4.5 ~ 5.0, injects water to 2500ml, aseptic filtration, and subpackage to obtain final product.
Prepared by embodiment 3 salvianolic acid L tablet
The preparation prescription of salvianolic acid L tablet:
Technique:
1 granulates
In salvianolic acid L and prescription, other adjuvant crosses 100 mesh sieves respectively, taking recipe quantity Radix Salviae Miltiorrhizae total phenolic acids extract adopts the equivalent method of progressively increasing to mix homogeneously with microcrystalline Cellulose, starch and carboxymethyl starch sodium, with appropriate PVP ethanol solution soft material, 14 mesh sieves are granulated, 50 ~ 60 DEG C of dryings 1 hour, add the magnesium stearate 14 mesh sieve granulate of recipe quantity.
2 tablettings
Get the special-shaped punch die tabletting of the special rhombus of above-mentioned granule.
The preparation prescription of embodiment 4 salvianolic acid L capsule:
Technique:
1 granulates
In salvianolic acid L and prescription, other adjuvant crosses 100 mesh sieves respectively, taking recipe quantity Radix Salviae Miltiorrhizae total phenolic acids extract adopts the equivalent method of progressively increasing to mix homogeneously with starch and carboxymethyl starch sodium, with appropriate PVP ethanol solution soft material, 14 mesh sieves are granulated, 50 ~ 60 DEG C of dryings 1 hour, add the magnesium stearate 14 mesh sieve granulate of recipe quantity.
2 fills
Get above-mentioned granule to incapsulate.
Claims (10)
1. salvianolic acid L or the application in preparation treatment or the Fibrotic medicine of prevention organ of its pharmaceutically acceptable salt, solvate and hydrolyzable ester.
2. application according to claim 1, described organ fibrosis is hepatic fibrosis or renal fibrosis.
3. application according to claim 1, described application is the increment that salvianolic acid L can suppress hepatic stellate cell.
4. application according to claim 1, described application is that salvianolic acid L salvianolic acid L can reduce Serum ALT, AST enzymatic activity.
5. application according to claim 1, described application is the content that salvianolic acid L can suppress liver Hyp.
6. application according to claim 1, described application is that salvianolic acid L can alleviate extent of liver fibrosis.
7. application according to claim 1, described application is that salvianolic acid L can suppress the deposition of III Collagen Type VI in liver.
8. application according to claim 1, described application is that salvianolic acid L can anti peroxidation of lipid.
9. application according to claim 1, described application is that salvianolic acid L has anti-liver cirrhosis, the effect of fatty liver and hepatic injury.
10. application according to claim 1, described medicine is using salvianolic acid L or its pharmaceutically acceptable salt, solvate and hydrolyzable ester as sole active agent or the pharmaceutical composition as one of active component.
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| CN2013104494078 | 2013-09-24 | ||
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1513848A (en) * | 2003-08-19 | 2004-07-21 | 江苏扬子江药业集团有限公司 | Method of extracting phenolic components from chinese medicine red sage root and its freeze dried powder injection agent |
| CN1951380A (en) * | 2005-10-21 | 2007-04-25 | 中国科学院上海药物研究所 | Medical usage of salvianolic acid B salt and total salvianolic acid |
| WO2010111935A1 (en) * | 2009-03-30 | 2010-10-07 | 天津天士力制药股份有限公司 | New salvianolic acid compound l, preparation method and use thereof |
| CN102475698A (en) * | 2010-11-29 | 2012-05-30 | 天津天士力制药股份有限公司 | Application of salvianolic acid L in preparation of medicines used for treating tumor |
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2014
- 2014-09-17 CN CN201410475966.0A patent/CN104434899A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1513848A (en) * | 2003-08-19 | 2004-07-21 | 江苏扬子江药业集团有限公司 | Method of extracting phenolic components from chinese medicine red sage root and its freeze dried powder injection agent |
| CN1951380A (en) * | 2005-10-21 | 2007-04-25 | 中国科学院上海药物研究所 | Medical usage of salvianolic acid B salt and total salvianolic acid |
| WO2010111935A1 (en) * | 2009-03-30 | 2010-10-07 | 天津天士力制药股份有限公司 | New salvianolic acid compound l, preparation method and use thereof |
| CN102475698A (en) * | 2010-11-29 | 2012-05-30 | 天津天士力制药股份有限公司 | Application of salvianolic acid L in preparation of medicines used for treating tumor |
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Address after: 300410 Tianjin city Beichen District Huaihe road and road intersection Dingjiang tianzhijiao Park forensic Center for Intellectual Property Department Applicant after: Tasly Pharmaceutical Group Limited by Share Ltd Address before: 300410 Tianjin city Beichen District Huaihe road and road intersection Dingjiang tianzhijiao Park forensic Center for Intellectual Property Department Applicant before: Tasly Pharmaceutical Group Co., Ltd. |
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Application publication date: 20150325 |