CN101181345A - Application of flos Sophora chromocor extract in the preparation of medicament for restrainting uric acid transporter URAT1 - Google Patents
Application of flos Sophora chromocor extract in the preparation of medicament for restrainting uric acid transporter URAT1 Download PDFInfo
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- CN101181345A CN101181345A CNA200710190133XA CN200710190133A CN101181345A CN 101181345 A CN101181345 A CN 101181345A CN A200710190133X A CNA200710190133X A CN A200710190133XA CN 200710190133 A CN200710190133 A CN 200710190133A CN 101181345 A CN101181345 A CN 101181345A
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- chromocor extract
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- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention relates to a new purpose of extract of charred flos sophorae immaturus, in particular to the application of the extract of the flos sophorae immaturus in the preparation of uricometer URAT1 suppressant drugs. The test on the animals shows that: the extract 1 and 2 of the flos sophorae immaturus has precise depressant effect on the hyperuricemia model animals to express the abnormally-increased uricometer URAT1, but has no remarkable depressant effect on the normal uricometer of normal animals, the safety is great. The extract 1 and 2 of the flos sophorae immaturus as well as relevant supplementary materials can be prepared into drugs for inhibiting the uricometer URAT1 by a regular preparation method and the drugs can be used for curing diseases relating the abnormally high expression of the uricometer URAT1.
Description
Technical field: the present invention relates to the application of flos Sophora chromocor extract aspect the preparation medicament for restraining uric acid transportor URAT 1.
Background technology: uric acid discharge to reduce or generates and increases, and can cause that uric acid concentration increases in the blood, directly or indirectly causes relevant disease.The drainage of uric acid is a complex process, and when uric acid flows into glomerule with blood circulation, the uric acid of free type will all filter; Heavily absorbed (Diamond HS, MeiselAD.Post secretory reabsorption of urate in man.Arthritis And Rheumatism, 1975,18 (6): 805-809.) in about 99% urate of near-end renal tubules.Urate transporter (albumen) URAT1 that is positioned at proximal convoluted tubule participates in the heavy absorption process of uric acid, by with anionic exchange urate being transported into cell by tube chamber.URAT1 albumen mainly is distributed in the renal cortex proximal tubular epithelial cells to the chamber film, is an electroneutral uric acid transporter albumen.The URAT1 gene is equaled at first to clone out from people's kidney in 2002 by Enomoto, be positioned at chromosome 11q13, by the SLC22A12 gene code, form by 9 exons of 10 introns, have 12 membrane spaning domains, its cDNA total length 2642bp, coding region 1659bp, coding contains 555 amino acid whose protein.Constitutional kidney Hypouricemia (hypouricemia) patient's the further reference's of SLC22A12 genetic flaw case URAT1 (hURAT1) participates in heavily absorption (the Atsushi Enomoto of uric acid, Hiroaki Kimura, ArthitChairoungdua, Yasuhiro Shigeta, et al.Molecular identification of arenal urate-anion exchanger that regulates blood urate levels.Nature, 2002,417 (6887): 447-452).Be rich in the natural flavone constituents in the Flos Sophorae Immaturus, can obtain high-load flavone extract by effective extraction separation purification refine step.
Summary of the invention
The object of the present invention is to provide the new purposes of flos Sophora chromocor extract, specifically provide the application of flos Sophora chromocor extract in the preparation medicament for restraining uric acid transportor URAT 1.
One of technical solution of the present invention: the Flos Sophorae Immaturus adopts alkali extraction and acid precipitation to extract, the adding distil water recrystallization, obtain flos Sophora chromocor extract respectively by the polydextran gel column separating purification obtain flos Sophora chromocor extract 1 and acid system hydrolysis, purification obtains flos Sophora chromocor extract 2.Quantitative through HPLC, flos Sophora chromocor extract 1 contains rutin 98%, and flos Sophora chromocor extract 2 contains Quercetin 98%.
Two of technical solution of the present invention: provide flos Sophora chromocor extract 1 and 2 to be used to prepare the new purposes of medicament for restraining uric acid transportor URAT 1.This new purposes has comprised with flos Sophora chromocor extract 1 of the present invention and 2, be equipped with the known pharmaceutic adjuvant of those skilled in the art (excipient, cosolvent, controlled release agent etc.), make the known dosage form of those skilled in the art (comprising oral liquid, injection, capsule, tablet, granule, microcapsule etc.), be used to prepare medicament for restraining uric acid transportor URAT 1.
Advantage of the present invention is, the flos Sophora chromocor extract 1 and 2 that urate transporter URAT1 is had definite inhibition regulating action is provided, and is used to prepare medicament for restraining uric acid transportor URAT 1, can be used for treatment and the unusual high expressed relevant disease of urate transporter URAT1.Compare with the invention at gout or antihyperuricemic disease, the new purposes of flos Sophora chromocor extract provided by the invention has 3 significantly progressive and advantages: 1, definite ingredients (being respectively flos Sophora chromocor extract 1 that contains rutin 98% and the flos Sophora chromocor extract 2 that contains Quercetin 98%); 2, action target spot clear and definite (the urate transporter URAT1 of Reabsorption in the urate excretion process); 3, regulating action clear and definite (suppressing to regulate).
The present invention utilizes the antihyperuricemic animal model scientifically to estimate on gene transcription level and protein expression level and has determined the inhibition regulating and controlling effect of flos Sophora chromocor extract to urate transporter URAT1.
The urate transporter URAT1 that the flos Sophora chromocor extract 1 involved in the present invention and the 2 pairs of animal pattern abnormal expressions increase has definite inhibitory action; And the urate transporter of intact animal's normal level is expressed no remarkable inhibitory action, have good safety.
Essence for a better understanding of the present invention below will be refining with the extraction of flos Sophora chromocor extract 1 and 2, pharmacological evaluation and result illustrate its application in the preparation medicament for restraining uric acid transportor URAT 1.
The specific embodiment: following examples are only as the usefulness of further setting forth invention, can not be used for limiting the present invention.
Embodiment 1: flos Sophora chromocor extract 1 and 2 preparation
Flos Sophorae Immaturus boiling water extraction (lime cream is transferred pH8 ~ 9), extracting solution is sucking filtration while hot, extracts repeatedly three times, merging filtrate, concentrated hydrochloric acid accent pH3 ~ 4, acid liquid is placed the dry back of crystallize sucking filtration precipitation and was added the water recrystallization by 1: 200, makes with extra care and obtains flavone extract.
Above-mentioned flavone extract polydextran gel column separating purification obtains flos Sophora chromocor extract 1.
Above-mentioned flavone extract is according to adding 2%H at 1: 100
2SO
4Boiled hydrolysis 2 hours-→ the cooling sucking filtration after washing residue to neutral, with mistake polyamide column chromatography purification behind 95% ethyl alcohol recrystallization, obtain flos Sophora chromocor extract 2.
Detect through the HPLC method: flos Sophora chromocor extract 1 contains rutin 98%
Flos Sophora chromocor extract 2 contains Quercetin 98%
Embodiment 2: flos Sophora chromocor extract 1 (containing rutin 98%) is to the inhibition regulating action of urate transporter URAT1
Laboratory animal: Kunming mouse, the 15-20 gram, male
The medicine preparation: flos Sophora chromocor extract 1 is scattered in the normal saline, is used for gastric infusion, and dosage is 25mg/kg
Experiment material: Trizol Reagment (Invigen), chloroform, isopropyl alcohol, dehydrated alcohol, DEPC, M-MLV reverse transcription, PCR test kit, RIPA lysate etc.
Experimental apparatus: High speed refrigerated centrifuge, high-speed homogenization machine, Bio-rad vertical electrophoresis groove, MBI-PCR instrument, horizontal strip electrophoresis groove etc.
Experimental model: mice hyperuricemia model.
Experimental technique:
1, the foundation of model: animal conformed after 1 week, irritated stomach and gave oxonic acid potassium salt 250mg/kg; Contrast gives equal-volume normal saline, 10 days modeling cycles.
2, administration: administration simultaneously during the modeling, after giving oxonic acid potassium salt modeling/normal saline contrast 1h, irritate stomach and give 25mg/kg flos Sophora chromocor extract 1; Contrast gives normal saline.
3, the preservation of mice execution and tissue: mice gives flos Sophora chromocor extract 1/ normal saline in the filling stomach and puts to death after 1 hour, separates renal tissue on the ice platform, and nephridial tissue is stored in-80 ℃ behind liquid nitrogen freezing.
4, western blotting (Western-blotting): get the RIPA lysate homogenate extraction that the about 100mg of tissue adds 1ml, 4 ℃ of 12000g are centrifugal 15 minutes, get the renal tissue total protein extracting solution, the Bradford method is surveyed protein concentration, and diluted protein sample to final concentration is 5ug/ul.Mix sample in the sample-loading buffer degeneration, PVDF changes film behind the SDS-PAGE gel electrophoresis, the confining liquid sealing adds mURAT1 antibody and (prepares according to ncbi database mURAT1 protein sequence after 1 hour, working concentration 1: 4000) 4 ℃ of overnight incubation, two anti-(1: 4000) room temperature shaking tables were hatched 1 hour, HRP-ECL is luminous, the darkroom exposure imaging.After the film scanning picture is carried out gray analysis.
5, RT-polymerase chain reaction (RT-PCR): extract the total RNA of mouse kidney, reverse transcription obtains the cDNA template, according to the mURAT1 gene design primer of ncbi database, carries out the amplification of mURAT1 genes of interest.Amplified production is the UV imaging behind agarose gel electrophoresis, and picture is carried out gray analysis.
Experimental result:
A. western blotting Western-Blotting
| Group n＝5 | Blank+normal saline | Blank+extract 1 | Model+normal saline | Model+extract 1 |
| Gray value | 60.7±18.0 | 56.4±13.9 | 133.1±22.9 +++ | 79.2±17.7 ** |
+++Compare with blank+normal saline group P＜0.001
*P＜0.01 and model+normal saline group filling in more continuous 10 days stomach gives oxonic acid potassium salt 250mg/kg modeling and causes URAT1 albumen high level expression in the mice nephridial tissue, and gray value reaches 133.1, is significantly higher than blank 60.7.The flos Sophora chromocor extract 1 that the filling stomach gives 25mg/kg can significantly suppress the proteic high level expression of URAT1 in the model mice nephridial tissue, and gray value is 79.2, near the blank level.Illustrate that flos Sophora chromocor extract 1 significantly suppresses the urate transporter URAT1 protein expression that animal pattern increases unusually.Irritate stomach and give that the URAT1 protein expression level does not make significant difference in 1 pair of blank mice nephridial tissue of flos Sophora chromocor extract, illustrate that the URAT1 albumen of 1 pair of mice nephridial tissue of flos Sophora chromocor extract normal level does not have remarkable inhibitory action, have safety preferably.
B. RT-polymerase chain reaction RT-PCR
| Group n＝5 | Blank+normal saline | Blank+extract 1 | Model+normal saline | Model+extract 1 |
| Gray value | 20.4±5.7 | 18.8±8.1 | 58.4±10.6 ++ | 35.2±9.1 * |
++Compare with blank+normal saline group P＜0.01
*P＜0.05 and model+normal saline group filling in more continuous 10 days stomach gives oxonic acid potassium salt 250mg/kg modeling and causes URAT1 mRNA high level expression in the mice nephridial tissue, and gray value reaches 58.4, is significantly higher than blank 20.4.Irritate flos Sophora chromocor extract 1 that stomach gives 25mg/kg and can significantly suppress the high level expression of URAT1 mRNA in the model mice nephridial tissue, gray value is 35.2, near the blank level.Illustrate that the urate transporter URAT1 mRNA that flos Sophora chromocor extract 1 can suppress to increase unusually expresses on transcriptional level.Irritate stomach and give that URAT1 mRNA expression is 18.8 in the blank mice nephridial tissue of flos Sophora chromocor extract 1, there is not significant change with respect to the blank group, the URAT1 mRNA that 1 pair of mice nephridial tissue of flos Sophora chromocor extract normal level is described does not have remarkable inhibitory action, has safety preferably.
Embodiment 3: flos Sophora chromocor extract 2 (containing Quercetin 98%) is to the inhibition regulating action of urate transporter URAT1
Experimental model and experimental technique are with embodiment 2, and flos Sophora chromocor extract 2 gastric infusion dosage are 25mg/kg
Experimental result:
A. western blotting Western-Blotting
| Group n＝5 | Blank+normal saline | Blank+extract 2 | Model+normal saline | Model+extract 2 |
| Gray value | 40.7±14.2 | 37.5±10.6 | 76.1±18.1 ++ | 55.2±14.2 * |
++Compare with blank+normal saline group P＜0.01
*P＜0.05 and model+normal saline group filling in more continuous 10 days stomach gives oxonic acid potassium salt 250mg/kg modeling and causes URAT1 albumen high level expression in the mice nephridial tissue, and gray value reaches 76.1, is significantly higher than blank 40.7.The flos Sophora chromocor extract 2 that the filling stomach gives 25mg/kg can significantly suppress the proteic high level expression of URAT1 in the model mice nephridial tissue, and gray value is 55.2, near the blank level.Illustrate that flos Sophora chromocor extract 2 significantly suppresses the urate transporter URAT1 protein expression that animal pattern increases unusually.Irritate stomach and give that the URAT1 protein expression level does not make significant difference in 2 pairs of blank mice nephridial tissues of flos Sophora chromocor extract, illustrate that the URAT1 albumen of 2 pairs of mice nephridial tissues of flos Sophora chromocor extract normal level does not have remarkable inhibitory action, have safety preferably.
B. RT-polymerase chain reaction RT-PCR
| Group n＝5 | Blank+normal saline | Blank+extract 2 | Model+normal saline | Model+extract 2 |
| Gray value | 29.6±6.7 | 27.5±8.9 | 48.1±12.6 + | 33.2±11.0 * |
+Compare with blank+normal saline group P＜0.05
*P＜0.05 and model+normal saline group filling in more continuous 10 days stomach gives oxonic acid potassium salt 250mg/kg modeling and causes URAT1 mRNA high level expression in the mice nephridial tissue, and gray value reaches 48.1, is significantly higher than blank 29.6.Irritate flos Sophora chromocor extract 2 that stomach gives 25mg/kg and can significantly suppress the high level expression of URAT1 mRNA in the model mice nephridial tissue, gray value is 33.2, near the blank level.Illustrate that the urate transporter URAT1 mRNA that flos Sophora chromocor extract 2 can suppress to increase unusually expresses on transcriptional level.Irritate stomach and give that URAT1 mRNA expression is 27.5 in the blank mice nephridial tissue of flos Sophora chromocor extract 2, there is not significant change with respect to the blank group, the URAT1 mRNA that 2 pairs of mice nephridial tissues of flos Sophora chromocor extract normal level is described does not have remarkable inhibitory action, has safety preferably.
Embodiment 4:
Flos Sophora chromocor extract 1 and 2 according to the conventional formulation method, is added entry and an amount of solubilizing agent (PEG400) dissolving, packing, sterilization, being prepared into specification is the flos Sophora chromocor extract oral liquid 1 and 2 of 40mg/ml;
According to the conventional formulation method, the soft capsule material is selected gelatin and sorbitol for use with flos Sophora chromocor extract 1 and 2, and being prepared into specification is the flos Sophora chromocor extract capsule 1 and 2 of 40mg/ grain;
Flos Sophora chromocor extract 1 and 2 according to the conventional formulation method, is added the excipient cyclodextrin, and mix homogeneously is granulated, tabletting, and being prepared into specification is the flos Sophora chromocor extract tablet 1 and 2 of 40mg/ sheet.
Claims (4)
1. the application of flos Sophora chromocor extract in the preparation medicament for restraining uric acid transportor URAT 1.
2. the application of flos Sophora chromocor extract according to claim 1 is characterized in that described flos Sophora chromocor extract is flos Sophora chromocor extract 1 and 2.
3. the application of flos Sophora chromocor extract according to claim 2 is characterized in that described flos Sophora chromocor extract 1 contains rutin 98%.
4. the application of flos Sophora chromocor extract according to claim 2 is characterized in that described flos Sophora chromocor extract 2 contains Quercetin 98%.
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