CN107951923A - A kind of preparation method of astragalus leaf flavonoids and its application in protection blood vessel drug eluting is prepared - Google Patents
A kind of preparation method of astragalus leaf flavonoids and its application in protection blood vessel drug eluting is prepared Download PDFInfo
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Abstract
The present invention relates to a kind of astragalus leaf flavonoids preparation method and its preparing the application in protecting blood vessel drug eluting, astragalus leaf will be dried and crush to be placed in 55% 65% ethanol and soak 4 7h, and be ultrasonically treated, then soak is filtered out.Seepage pressure effects are carried out with 8 times of volumes of medicinal material amount, 55% 65% ethanol again, and soak and percolate are concentrated under reduced pressure into no alcohol taste, then using water dilute liquid medicine concentration as 0.25g/ml containing crude drug.Add ethyl acetate to extract 3 times, combining extraction liquid, which is evaporated, to get dry extract.Dry cream is diluted to 15 25mg/ml with water dissolving, by 8 type large pore resin absorption columns of AB, with 55% ethanol elution.Be concentrated under reduced pressure dry astragalus leaf flavonoids extract yield >=20%.This method can improve recovery rate and shorten extraction time, and concentration of alcohol used in diacolation reduces, and the astragalus leaf flavonoids of preparation have the function that to protect blood vessel, realize that Engelhardia roxburghina Wall.total flavones are preparing the application in protecting blood vessel drug eluting.
Description
Technical field
Blood vessel drug eluting is protected the present invention relates to medicine, the particularly a kind of preparation method of astragalus leaf flavonoids and its preparing
In application.
Background technology
Diabetes are a kind of chronic metabolic obstacle diseases, its illness rate, disability rate and case fatality rate occupy in chronic disease
Three.China patient numbers have ranked the first in the world.At the beginning of cut-off 2011, in China 18 years old and above crowd's population sample, estimation
Diabetes morbidity is 11.6%, there are about 1.139 hundred million patients.The year two thousand thirty is expected, global diabetic's number will be increased to
5.52 hundred million people.
Diabetes are divided into 1 type and 2 types again, and the pathogenesis of wherein diabetes B (T2DM) is mainly reflected in insulin and supports
Anti- and defect of insulin secretion.At present, the cardiovascular risk of diabetes B chronic vascular complications is very big, 70-80% glycosurias
Patient's major causes of death be not diabetes in itself, but atherosclerotic cardiovascular disease (ASCVD) complication,
Such as coronary heart disease, cerebral arterial thrombosis and peripheral arterial disease.At present, domestic and international Guidelines for Management of Diabetes Mellitus is recommended, for
T2DM patient, it is necessary to strengthen the integrated management of the multiple cardiovascular risk factors such as hyperglycaemia, hypertension, dyslipidemia, obesity,
To reduce cardiovascular event and mortality risk to greatest extent.Therefore, for merging the T2DM patient of ASCVD, except hypoglycemic
Treatment, should also be aided with the treatment such as lipid-loweringing, decompression, and Comprehensive management is carried out to this kind of patient.
The hypoglycemic medicine of clinical treatment T2DM mainly has biguanides, sulfonylureas, thiazolidinedione medicine, non-sulfonylurea at present
Class insulinotropic hormone excretion, glucagon-like-peptide-1 (GLP-1) analog and DPP IV (DPP-4) inhibitor
Deng.Its therapy mechanism is respectively：(1) biguanides：Promote uptake and utilization of the tissue to glucose, suppress hepatic gluconeogenesis, promote
The uptake and utilization of the peripheral tissues to glucose such as the anerobic glycolysis of sugar entering, increase muscle.Representing medicine has melbine etc..
(2) sulfonylureas：Stimulate insulin secretion；Representing medicine has glibenclamide, gliclazide, gliquidone etc..(3) glucose sugar
Glycosides enzyme inhibitor：The Reverse transcriptase glucuroide in intestines and stomach, delays disaccharide in enteric cavity, oligosaccharide and polysaccharide release Portugal
Grape sugar, finally reduces postprandial blood sugar.Representing medicine has acarbose, Miglitol etc..(4) thiazolidinedione medicine：Insulin
Sensitizer, by increase peripheral tissues to the sensitiveness of insulin, improve insulin resistance and reduce blood glucose.Medicine is represented as sieve
Lattice row ketone, Pioglitazone etc..(5) non-sulfonylureas insulinotropic hormone excretion：By blocking potassium ion on beta Cell of islet film to lead to
Road, so as to promote insulin secretion.Medicine is represented as Repaglinide, Nateglinide etc..(6) glucagon-like-peptide-1 (GLP-
1) analog：Glucose dependency insulin secretion accelerating.Medicine is represented as Liraglutide, Exenatide etc..(7) dipeptidyl peptidase
Enzyme IV (DPP-4) inhibitor：Suppress DPP-4 enzymatic activitys, enhancing glucagon-like-peptide-1 (GLP-1) and Gastric inhibitory polypeptide activity, into
And promote insulin secretion.Medicine is represented as sitagliptin.
There is multinomial clinical data to show, in numerous antidiabetic drugs, only a small number of medicines clearly have cardiovascular benefit, such as
Melbine, Liraglutide.For the diabetes B patient for ASCVD complication occur, simple doctor trained in Western medicine hypoglycemic medicine
The cardiovascular curative effect that can possess is often limited.Therefore it is a kind of to have hypoglycemic, lipid-loweringing and the traditional Chinese medical science drop for protecting vascular function concurrently
Sugared medicine certainly will become a kind of Superior selection for the treatment of diabetes B ASCVD complication.
Theory of traditional Chinese medical science thinks, the basic pathogenesis of diabete (diabetes) be YIN fluid deficiency, it is scorching contain partially, and using the deficiency of Yin as
Originally it is, scorching for mark.It is mainly for the clinical treatment feature that the interpretation of the cause, onset and process of an illness is taken：Supplementing qi and nourishing yin, strengthening spleen, tonifying kidney, rush down clearly latent heat, work
Blood stagnation resolvation.We have found in laboratory by the research to Chinese medicine medication experience among the people, clearing heat and detoxicating and promoting blood circulation and removing blood stasis to diabete
Treatment has good improvement result.
Astragalus leaf is the dried leaf of Juglandaceae astragalus platymiscium astragalus Engelhardia roxburghiana Wall, is had
There is the effect of clearing heat and detoxicating, quench one's thirst of promoting the production of body fluid, relieving summer-heat dampness removing, for treating the swollen vexed, cat fever of taste humidity hysteresis, chest and abdomen, damp and hot letting out
Rush down, hernia abdominal pain etc..The components such as chromocor compound, steroid and triterpenes are mainly contained in astragalus leaf.Astragalus Ye is always yellow at present
The extracting method of ketone, which has been shown in, has part to disclose report, such as decocting, cold soaking, the method such as simple diacolation, and not only general flavone extract yield
Low, extraction time length, the concentration of alcohol of used extraction is also higher, and ultimate cost is also higher.
The content of the invention
For the above situation, to overcome the defect of the prior art, it is always yellow that the purpose of the present invention is just to provide a kind of astragalus Ye
The preparation method of ketone, it is low effectively to solve astragalus leaf flavonoids extract yield in the prior art, and the time is long and ethanol is extracted into
The problem of this is high.
Technical proposal that the invention solves the above-mentioned problems is to crush dry astragalus leaf and be placed in 55%-65% ethanol
4-7h is soaked, is ultrasonically treated, then filters out soak at the same time.Again with the 55%-65% ethanol of 8 times of volumes (ml) of medicinal material amount
Seepage pressure effects are carried out, obtain percolate.It is preferred that during whole immersion and diacolation, it is ultrasonically treated in extraction vessel.
Gained soak and percolate are finally concentrated under reduced pressure into no alcohol taste, then diluted with water, adjustment liquor strength is equivalent to containing life
Medicine 0.25g/ml.The ethyl acetate extraction of same volume is added, repeats extraction 3 times, 3 extracts of merging, which are evaporated, to get dry extract.Will be dry
It is about 15-25mg/ml that cream is diluted to mass concentration with water dissolving, and AB-8 type large pore resin absorption columns in sample liquid, are washed with ethanol
It is de-, obtain eluent.Ethanol is recovered under reduced pressure in eluent, is concentrated and dried, obtains astragalus leaf flavonoids.Surveyed through ultraviolet spectrophotometry
It is fixed, astragalus leaf flavonoids extract yield >=20%.The astragalus leaf flavonoids protect blood vessel drug eluting effective for preparing, and realize astragalus
General flavone is preparing the application in protecting blood vessel drug eluting.
Preparation method of the present invention is simple, abundant raw materials, and ultrasonic wave can damage cells wall, increases solvent penetration power, passes through
Combined with percolation, recovery rate can be improved and shorten extraction time, thus efficiently, quick prepare astragalus leaf flavonoids.Ooze at the same time
Concentration of alcohol used of filtering is reduced compared with the concentration that the prior art is reported, can also be achieveed the purpose that cost-effective.The astragalus Ye of preparation is total
Flavones has the function that to protect blood vessel, protects blood vessel drug eluting effective for preparing, opened up treatment diabetic cardiovascular complications
New application.
Embodiment
Elaborate with reference to following concrete condition to the embodiment of the present invention.
Embodiment 1：Dry astragalus leaf is crushed to be placed in 55% ethanol and soaks 5h, then filters out soak.Then
Seepage pressure effects are carried out with 55% ethanol of 10-15 times of volume of medicinal material amount (ml) again, diacolation speed is 18ml/ (min.kg), must be oozed
Filter liquid.During whole immersion and diacolation, high-power focusing ultrasonic rod is inserted into extraction vessel and is carried out at ultrasound
Reason, finally by gained soak and percolate, is concentrated under reduced pressure into no alcohol taste, then dilute with water, adjustment liquor strength be equivalent to
0.25g/ml containing crude drug.The ethyl acetate extraction of same volume is added, repeats extraction 3 times, 3 extracts of merging, which are evaporated, to get dry extract.
It is about 20mg/ml that dry cream is diluted to mass concentration with water dissolving, AB-8 type large pore resin absorption columns in sample liquid, applied sample amount
Ratio with resin volume is 1: 5, and resin column blade diameter length ratio is 1: 10；With 55% ethanol elution of 8 times of column volumes, eluent is obtained.
Ethanol is recovered under reduced pressure in eluent, is concentrated and dried, obtains astragalus leaf flavonoids.Through determined by ultraviolet spectrophotometry astragalus leaf flavonoids
Extract yield >=20%.The astragalus leaf flavonoids protect blood vessel drug eluting effective for preparing, and realize that Engelhardia roxburghina Wall.total flavones are preparing guarantor
Protect the application in blood vessel drug eluting.
Comparative example：Take astragalus leaf to crush, 70% ethanol immersion 8h is added, after filtering out leachate, with 16 times of medicinal material amount
70% ethanol percolation.No alcohol taste is concentrated under reduced pressure into percolate, after being diluted with water, with ethyl acetate abstraction impurity removal, repeats to extract
3 times.3 extracts are evaporated and are got dry extract.By dry cream through macroporous resin purification, with 50% ethanol elution, final recycling gained is yellow
Qi general flavone.It is 14% through general flavone yield obtained by determined by ultraviolet spectrophotometry.
Preparation method of the present invention is simple, abundant raw materials, and it is always yellow that ultrasound is combined the more efficient preparation astragalus Ye of energy with percolation
Ketone, when length, and the Extraction solvent concentration used accordingly reduces, and can save manufacturing cost.The astragalus leaf flavonoids of preparation have
Hypoglycemic, lipid-loweringing, the effect for protecting blood vessel, blood vessel drug eluting is protected effective for preparing, and satisfied effect is obtained through experiment, is had
It is as follows to close experimental data：
1. pair diabetes B merges the influence of atherosclerotic rat model
1. experiment material
1.1 experimental animals and feed
It is dynamic that cleaning grade male Goto-Kakisaki (GK) rats and SPF grades of Wistar rats are purchased from Shanghai Si Laike experiments
Thing Co., Ltd, animal credit number SCXK (Shanghai) 2012-0002.
1.2 medicines and reagent
Astragalus leaf, which is picked up from Guangxi, to be thought, and astragalus dried leaf is accredited as through Guangxi traditional Chinese medicine research institute Chinese medicine.Astragalus Ye is total
Flavones is extracted by Guangxi traditional Chinese medicine institute and prepared, and carries out Quality Control；Your treasured system metformin hydrochloride tablet, Sino-U.S. apply in Shanghai
Medicine Co., Ltd, lot number AA62812；Atorvastatin, pfizer inc, lot number L80727；Cholesterol, pig
Courage powder, propylthiouracil (PTU), L-NAME, are purchased from Shanghai Yuan Ye bio tech ltd；Noradrenaline bitartrate
(NE), Shanghai Hefeng Pharmaceutical Co., Ltd.；Chloraldurate, Sinopharm Chemical Reagent Co., Ltd.；HDL, LDL, TG, TC are surveyed
Box is tried, Nanjing is purchased from and builds up Bioengineering Research Institute；Haematoxylin Yihong (HE) staining kit, the examination of Masson trichrome stains
Agent box, is purchased from Beijing Lei Gen Bioisystech Co., Ltd；RNA extraction agents box, PrimeScript reverse transcription reagent box,
SYBR quantification kits, Ago-Gel, are purchased from Japanese Takara Bio Inc.Rat-actin primers, upstream
5 '-GGAGATTACTGCCCTGGCTCHCTA-3 ', downstream 5 '-GACTCHATCHGTACTCHCTGCTTGCTG-3 '；Rat NF- κ
B p65 primers, upstream 5 '-CGACGTATTGCTGTGCCTTCH-3 ', downstream 5 '-TTGAGATCHTGCCCAGGTGGTA-3 '；
Rat ICAM-1 primers, upstream 5 '-GCTTCHTGCCACCATCHACTGTGTA-3 ', downstream 5 '-
ATGAGGTTCHTTGCCCACCTG-3 ', above primer are synthesized by precious bioengineering (Dalian) Co., Ltd.
1.3 laboratory apparatus
Accu-Chek Active blood glucose meters, Roche Holding Ag of the U.S.；Biological function pilot system, Chengdu Tai Meng science and technology are limited
Company；High speed freezing centrifuge, German Hettich Scientific Instruments Corporations；Multiskan GO microplate reader, the silent winged generation of U.S.'s match
You are scientific and technological；The ultraviolet gel imagers of Gel Doc XR+, Bio Rad companies；7300 real-time fluorescence quantitative PCR instrument, the U.S.
Applied Biosystems companies；Horizontal cataphoresis apparatus, Liuyi Instruments Plant, Beijing；Paraffin wax embedding, histotome, optics
Microscope, Leca company.
2. experimental method
2.1 diabetes Bs merge atherosclerotic rat model foundation
Male GK rats are taken, high lipid food is given and feeds 4 weeks, while on day 1 to the 14th day intraperitoneal injection nitric oxide
Synthase (NOS) inhibitor L-NAME (5mg/kg), atherosclerosis-inducing formation.Fasting blood was measured at the end of the 4th week
Sugar, takes the GK rats of 11.1 mmol/L of fasting blood sugar ＞ to be tested.
2.2 packets and administration
Standard compliant rat is randomly divided into model group, melbine group (150mg/ by fasting blood glucose level by more than
Kg), Atorvastatin group (10mg/kg), astragalus leaf flavonoids high and low dose group (500,250mg/kg), every group 8.Separately take
8 normal Wistar rats are set to control group, control rats feeding normal diet, remaining group continues to be fed with high lipid food.Point
After group, each group gavage gives corresponding dosage medicine 8 weeks, and control group gives equivalent distilled water with model group gavage.
2.3 serological index measure
Rat-tail hematometry each group rat fasting blood-glucose is adopted at administration the 2nd, 4,6,8 weekends, abdominal aorta is taken in the 8th weekend
Blood, centrifugation measure serum hdl, LDL, TG, TC are horizontal.
2.4 aorta pectoralis pathologic findings
8th weekend took part aorta pectoralis, hematocele in sustainer surrounding tissue and lumen of artery was removed on ice, through 4% poly
Formaldehyde is fixed, is dehydrated, is transparent, paraffin embedding and section, by HE staining kits and Masson trichrome stain kit description lines
HE is dyed and Masson dyeing, observes rat chest aorta pathological change.
2.5 aorta pectoralis NF- κ B p65 and ICAM-1 mRNA are detected
8th weekend took part aorta pectoralis, removes hematocele in sustainer surrounding tissue and lumen of artery on ice, sustainer is put
Enter and preserved in RNA liquid, in case total serum IgE extracting and detection.Negative control is done with normal structure, β-actin are as internal reference, with 2^
(- Δ Δ CT) method progress relative quantitative assay.
2.5.1 cDNA synthesis sterilizing in enzyme EP pipes without sequentially adding thoracic aortic tissue extract total serum IgE, gDNA
Eraser, 5gDNA Eraser Buffer and Rnase Free dH2O, 42 DEG C of 2min, 4 DEG C of holdings, remove genomic DNA.
Sterilizing without enzyme EP pipes in sequentially add above-mentioned reaction solution, PrimeScript RT Enzyme Mix1, RT primer Mix,
5primescript Buffer 2, Rnase Free dH2O, 37 DEG C of 15min, 85 DEG C of 5s, 4 DEG C of holdings, it is anti-to carry out reverse transcription
Should.
2.5.2 PCR reactions sequentially add reverse transcription reaction liquid, SYBR Premix Ex Taq in sterilizing is without enzyme EP pipes
II、PCR F-Primer、 PCR R-Primer、ROX Reference Dye、Rnase Free dH2O.Using two-step method into
Row PCR amplification, β-actin, the reaction condition of NF- κ B p65 and ICAM-1 are：95 DEG C of 30s pre-degenerations, 95 DEG C of 5s, 60 DEG C
31s expands 40 circulations.
2.6 in vitro aorta pectoralis systolic and diastolic function detections
Take model group rat, carefully wins aorta pectoralis, is placed in and fills in the ice-cold K-H liquid plate of logical O2 in advance, removes
Endovascular hematocele, separates and removes blood vessel peripheral adipose and connective tissue, and it is cut into the arterial ring of 3 ± 4mm, passes through
Upper and lower wire hanger connection tonotransducer, accesses biological functional system.The preload for giving arterial ring 1g is put down
Weighing apparatus, with the pre- vasoconstrictions of 60mmol/L KCl, tension variation is using blood vessel in more than 1g, is started in fact after being eluted to baseline
Test.Investigate in the case of NE (1 10-5mol/L) is pre-shrunk, astragalus leaf flavonoids (6.7 × 105mg/mL、2×104mg/
mL、6×104mg/mL、1.8×103mg/mL、5.4×103mg/mL、1.62×102Mg/mL) the easypro contracting to myocardium vessel is made
With control group gives normal saline.
2.7 statistical method
Data represent that data carry out statistical disposition using 16.0 softwares of SPSS with x ± s.Multigroup sample average compares two-by-two
Examined compared with using one-way analysis of variance, homogeneity of variance using Dunnett, heterogeneity of variance uses non-parametric test.Two groups independent
Sample average compares to be examined using Independent Sample T Test.P ＜ 0.05 are statistically significant for difference.
3 results
3.1 astragalus leaf flavonoids merge diabetes B the influence of the blood glucose and blood fat of atherosclerotic rat
Model group rats blood glucose rise is obvious, significant difference (the P ＜ 0.01) compared with control group.It is administered the 2nd week to the 8th
Week, melbine, astragalus leaf flavonoids high dose group blood glucose decline obvious, there were significant differences compared with model group (P ＜ 0.01
~0.05).It is administered the 4th week to the 8th week, compared with model group, astragalus leaf flavonoids low dose group blood glucose conspicuousness declines (P ＜
0.01) table 1, is referred to.It was administered for the 8th weekend, model group rats serum hdl, LDL and TC substantially increase, the difference compared with control group
Significantly (P ＜ 0.01).Compared with model group, Atorvastatin, astragalus leaf flavonoids are high and low dosage can significantly reduce rat serum
Clear LDL, TG and TC are horizontal (P ＜ 0.01~0.05).
3.2 astragalus leaf flavonoids merge atherosclerotic rat aorta pectoralis pathological change to diabetes B to be influenced
HE dyeing displays, model group rats aorta pectoralis theca interna are presented certain swelling, and with endothelium is exposed and fiber
It is disorganized to wait pathological change.Masson dyeing discloses, film layer collagenous fibres abnormality proliferation in model group aorta pectoralis.Administration 8
Zhou Hou, astragalus leaf flavonoids high and low dose group can significantly improve the swelling of aorta vessel inner membrance and collagenous fibres abnormality proliferation
Deng pathological change.
3.3 astragalus leaf flavonoids to diabetes B merge atherosclerotic rat aorta pectoralis NF- κ B p65 and
The influence of ICAM-1 mRNA level in-sites
Compared with control group, model group aorta pectoralis NF- κ B p65 and ICAM-1 mRNA expression dramatically increase；With mould
Type group is compared, melbine, Atorvastatin, astragalus leaf flavonoids height and low dose group NF- κ B p65 and ICAM-1 mRNA
Express equal conspicuousness and lower (P ＜ 0.01~0.05).
3.4 astragalus leaf flavonoids merge the in vitro aorta pectoralis systolic and diastolic function of atherosclerotic rat to diabetes B
Influence compared with control group, when addition final concentration of 1 × 105After mol/L NE shrink peaking in advance, according to cumulative concentration method according to
It is secondary addition astragalus leaf flavonoids up to final concentration be respectively 6.7 × 105mg/mL、2×104mg/mL、6×104mg/mL、1.8×
103mg/mL、5.4×103mg/mL、 1.62×102Mg/mL, it is found that astragalus leaf flavonoids above final concentration can substantially relax
Open the vascular contractile response mediated by NE.
Claims (4)
1. a kind of preparation method of astragalus leaf flavonoids, it is characterised in that dry astragalus leaf is crushed and is placed on 55%-65% second
4-7h is soaked in alcohol, is ultrasonically treated at the same time, then filters out soak, then with the 55%-65% ethanol of 8 times of volumes of medicinal material amount into
Row seepage pressure effects, obtain percolate, and gained soak and percolate then are concentrated under reduced pressure into no alcohol taste, then are diluted with water, adjustment
Liquor strength is 0.25g/ml containing crude drug, then adds the ethyl acetate extraction of same volume, repeats extraction 3 times, merges 3 extractions
Liquid, which is evaporated, to get dry extract, and the dry cream is diluted to mass concentration as 15-25mg/ml using water dissolving, AB-8 types macropore in sample liquid
The ratio of adsorption resin column, applied sample amount and resin volume is 1: 5, and resin column blade diameter length ratio is 1: 10；With 55% second of 8 times of column volumes
Alcohol elutes to obtain eluent, and the eluent is recovered under reduced pressure ethanol, is concentrated and dried, obtains astragalus leaf flavonoids, yield >=20%.
2. preparation method as claimed in claim 1, it is characterised in that be ultrasonically treated with during diacolation soaking.
3. preparation method as claimed in claim 1, it is characterised in that crush to be placed in 55% ethanol by dry astragalus leaf and soak
Steep 5h.
4. using astragalus leaf flavonoids prepared by any one of claim 1-3 the method answering in protection blood vessel drug eluting is prepared
With.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114796308A (en) * | 2021-12-14 | 2022-07-29 | 成都大学 | Application of engelhardtia roxburghiana leaf extract as alpha-glucosidase inhibitor |
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| CN1724552A (en) * | 2005-07-08 | 2006-01-25 | 南京大学 | Preparation method of astilbin |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114796308A (en) * | 2021-12-14 | 2022-07-29 | 成都大学 | Application of engelhardtia roxburghiana leaf extract as alpha-glucosidase inhibitor |
| CN114796308B (en) * | 2021-12-14 | 2024-03-19 | 成都大学 | Application of engelhardtia leaves extract as alpha-glucosidase inhibitor |
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Application publication date: 20180424 |