CN105106265A - Application of burdok root oligosaccharide in preparation of antithrombotic drug - Google Patents
Application of burdok root oligosaccharide in preparation of antithrombotic drug Download PDFInfo
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Abstract
The invention discloses novel application of burdok root oligosaccharide. Specifically, antithrombotic effect of burdok root oligosaccharide is discovered, so application of burdok root oligosaccharide in preparation of an antithrombotic drug is further provided in the invention. The burdok root oligosaccharide provided by the invention has low extraction cost and small toxic and side effects, can inhibit platelet aggregation and protect vascular endothelial cells, exerts antithrombotic effect and is applicable to preparation of the antithrombotic drug.
Description
Technical field
The present invention relates to a kind of purposes of Radix Arctii oligosaccharide, the application in antithrombotic reagent prepared by especially a kind of Radix Arctii oligosaccharide.
Background technology
Thrombotic disease (thromboembolicdisease) has been the frequently-occurring disease of serious harm human health at present, can be divided into phlebothrombosis and arterial thrombus.Arterial thrombus is because vascular endothelial cell is impaired mostly, platelet adhesion and gathering and cause; And phlebothrombosis is generally cause because blood flow is slow or the stasis of blood is stagnant.Fructus Arctii (ArctiumLappaL.) is Compositae 2 years raw herbaceous plant, and Radix Arctii contains various active composition, as Polyphenols, and aldehydes, polyacetylene class, several amino acids and sugar etc.The existing relevant report extracting Radix Arctii polysaccharide and oligosaccharide from Radix Arctii in recent years, research shows that burdock polysaccharide can regulate immunity of organisms, and in antitumor, anti-hepatitis, blood fat reducing, defying age etc. in have unique physiological function.But, the relevant report also do not applied in antithrombotic about Radix Arctii oligosaccharide up to now.
Summary of the invention
The present invention has found that Radix Arctii oligosaccharide has antithrombotic effect, and then has invented burdock root oligosaccharide and preparing the application in antithrombotic reagent.
Technical solution of the present invention is: the application in antithrombotic reagent prepared by a kind of Radix Arctii oligosaccharide.
Specifically preparing the application in anticoagulant medicine.
Application specifically in preparation protection vascular endothelial cell medicine.
Radix Arctii oligosaccharide extraction cost of the present invention is low, toxic and side effects is little, has anticoagulant, protection vascular endothelial cell and antithrombotic effect, can be applicable to prepare antithrombotic reagent.
Accompanying drawing explanation
Fig. 1 is embodiment of the present invention Radix Arctii oligosaccharide water extract SephadexG-50 elution profile.
Fig. 2 is that embodiment of the present invention HPGPC method high performance liquid chromatograph detects Radix Arctii oligosaccharide chromatogram.
Fig. 3 is the platelet aggregation rate design sketch under each moment point of each experimental group of Radix Arctii oligosaccharide of the present invention.
To be Radix Arctii oligosaccharide of the present invention damage the NO level of HY926 cell to rhTNF-α to Fig. 4 affects schematic diagram.
Fig. 5 is the effect schematic diagram of Radix Arctii oligosaccharide on Carrageenan induced mice arteria caudalis thrombosis average relative length of the present invention.
Fig. 6 Radix Arctii of the present invention oligosaccharide rat carotid artery of inducing FeCl3 is thrombotic affects schematic diagram.
Fig. 7 Radix Arctii oligosaccharide of the present invention affects schematic diagram to the rat platelet aggregation rate that ADP induces.
Fig. 8 Radix Arctii oligosaccharide of the present invention affects schematic diagram to AT-in rat plasma III content.
Detailed description of the invention
The preparation of Radix Arctii oligosaccharide:
Extracting method is as follows:
1) Radix Arctii is cleaned section, be placed in 60 DEG C of baking oven more than inner drying 12h, mechanical activation comminution, obtained dry powder;
2) taking step 1) gained Radix Arctii dry powder 20g is placed in 250ml round-bottomed flask, measure 100ml(4 ~ 5 times volume) 80% ethanol (ethanol: distilled water=4:1), 60 DEG C of reflux, extract, 6h, discard extracting solution to remove the alcohol dissolubility impurity such as monosaccharide and disaccharide, aglycon, solvent are volatilized to obtain dry powder;
3) measure 4 ~ 5 times of volume (100ml) petroleum ether pour into step 2 is housed) in the flask of gained Radix Arctii dry powder, after 45 DEG C of reflux, extract, 1.5h, solution is volatilized, obtained dry powder;
4) step 3) gained dry powder is poured in 1000ml flask, measures 300ml(15 doubly) distilled water adds in flask, 95 DEG C of heating in water bath 3h, and repeatedly extract four times, merging filtrate is concentrated into appropriate volume, obtains Radix Arctii oligosaccharide concentrated solution;
5) in step 4) gained concentrated solution, the Savage reagent (chloroform: n-butyl alcohol=4:1) of 1/5 volume is added, jolting 10min, centrifugal (3600rmin-1,15min), get supernatant, 4 ~ 5 times repeatedly, with fully except Deproteinization, obtain the upper water solution being rich in Radix Arctii oligosaccharide;
6) in step 5) gained supernatant, the dehydrated alcohol of 4 times of volumes is added, hold over night (>12h), centrifugal (5000rmin-1,15min), abandon supernatant, by precipitation 45 DEG C of water dissolutioies, precipitate with ethanol water extraction 4 ~ 5 times repeatedly, makes Radix Arctii oligosaccharide fully precipitate; Precipitation is respectively washed 3 times with dehydrated alcohol and acetone respectively, volatilizes solvent, lyophilization, obtain defat Deproteinated Radix Arctii oligosaccharide dry powder, for subsequent use;
7) the Radix Arctii water extract dry powder of gained is above measured its oligosaccharide content by sulphuric acid-phynol method, the oligosaccharide content of Radix Arctii water extract dry powder is 70%.
The purification process of the Radix Arctii water extract of gained is as follows:
1) sephadex G will newly bought
5020g distilled water rinsing 3 ~ 5 times, the gel particle floating over upper strata to be discarded, then through boiling water bath 1h, make it fully swelling and play the effect of sterilizing, then gel is loaded in chromatographic column (φ 2.0cm*60cm), then with the water of 2 ~ 3 volumes, post bed is stablized;
2) obtained refining Radix Arctii polyoses extract powder distilled water is dissolved (dissolving of 0.5g dry powder 5ml distilled water) with suitable dissolubility ratio, by obtained pharmaceutical aqueous solution through polydextran gel column chromatography;
3) sephadex chromatography condition is: take water as eluent, room temperature condition (25 DEG C), and flow velocity is 1.0ml/min ~ 2.0ml/min, automatic portion collection eluent, and every 2ml mono-manages;
4) detect by pipe with sulphuric acid-phynol method, light absorption value is measured at 492nm wavelength place by microplate reader, figure is with absorbance according to elution volume (or the pipe number collected), obtain corresponding elution profile (see accompanying drawing 1), eluent is merged according to the eluting peak in the elution profile obtained, concentrating under reduced pressure, obtains Radix Arctii oligosaccharide just pure products;
5) by concentrated solution by 1) ~ 4) purification again, concentrated, collect.
The Radix Arctii oligosaccharide dry powder of extraction purification gained is as stated above measured purity and molecular weight by HPGPC method, detects its chemical constitution by proton nmr spectra.
HPGPC method measures purity and molecular weight:
1) sample tri-distilled water is made into 1.5mg/ml solution;
2) Waters1525 high performance liquid chromatograph (ShodexOhpakSB-803HQ, 8.0mmI.D. × 300mm, Waters2410 differential refraction detector) detects, its testing conditions is: column temperature is 30 DEG C, mobile phase is tri-distilled water, and sample size is 20 μ l, and flow velocity is 0.8ml/min.Obtain high-efficient liquid phase chromatogram (see accompanying drawing 2);
3) according to peak area ratio calculation sample purity under main peak peak shape and different retention time.Adopt HPGPC method, a standard DextranT series glucosan is standard substance, calculates Mw.According to HPLC collection of illustrative plates, this extract is the Radix Arctii oligosaccharide that purity can reach 97.7%, and Mw is 2135Da.
Prepared Radix Arctii oligosaccharide is following experiment material.
Experiment:
Experimental example 1 Radix Arctii oligosaccharide is to the inhibitory action of the platelet aggregation that external ADP induces
1.1 experiment material
New zealand white rabbit, male, body weight 1.5 ~ 2kg, is provided by Dalian Medical Univ's animal center.
ADP powder is provided by the raw company of Shanghai Puli.
1.2 experimental technique
1.2.1 grouping is tested
The platelet aggregation model group that model group: ADP induces, not dosing.
Experimental group: the experimental group of point three dosages, adds the medicine of variable concentrations respectively; Add the ADP solution with model group same volume again.
1.2.2 experimental technique
New zealand white rabbit heart left ventricle obtains arterial blood, with 3.8% sodium citrate and blood in the ratio anticoagulant of 1:9, will obtain blood and anticoagulant mixing gently rapidly, avoids acutely shaking to prevent haemolysis.Anticoagulation is put into horizontal centrifuge first with the centrifugal 5min of the rotating speed of 800rmp, collect the milky blood plasma in upper strata and be platelet rich plasma (plateletrichplasma, PRP), by lower floor's blood cell fraction again through the centrifugal 5min of 2000rmp, get the faint yellow blood plasma fractions in upper strata and obtain platelet poor plasma (plateletpoorplasma, PPP).Adopt turbidimetry to carry out Platelet mensuration, utilize LBY-NJ2 type four-way platelet aggregation instrument to trace curve of platelet aggregation, maximum platelet aggregation rate in record 5min.
1.3 experimental result
Final concentration is that the ADP of 7.5 μ g/ml acts on PRP300s, obtain 60s, 180s, 300s and max platelet rate (max) respectively, can obtain according to the value of time point the time suppression ratio figure (see accompanying drawing 3) that a medicine changes platelet aggregation, the average maximum agglutination rate of each group sees the following form 1.
Table 1 Radix Arctii water extract is to the inhibitory action (x ± semn=10) of the platelet aggregation that ADP induces
Note: represent that each group compared with damage group:
*p≤0.05.
The protective effect that experimental example 2 Radix Arctii oligosaccharide damages human umbilical vein endothelial cell
In vascular endothelial cell, under quiescent condition, the eNOS catalysis in endotheliocyte produces the NO of low concentration, plays physiological regulating action, and protection endotheliocyte is to maintain the integrity of endothelium; When endothelium is upset, as in inflammation damnification situation, will activate iNOS, and then catalysis produces the NO of high concentration, bioavailability also reduces, and reacts with superoxide anion and generate harmful substance, and then endothelial cell injury function.This experiment, using NO content as the index of endothelial cell damage, meets existing theory and clinical study results.
1. experiment material
HY926 cell strain (human umbilical vein endothelial): presented by Department Of Medicine, Peking University's physiology and pathophysiology system professor Zhu Yi; DMEM dehydrated medium: Gibco company of the U.S.; Trypsin, hyclone (FBS): Tianjin Hao sun biotechnology company; Recombination human tumor necrosis factor-alpha (rhTNF-α): PEPROTECHAsia company of the U.S.; Nitric oxide (NO) test kit: Bioengineering Research Institute is built up in Nanjing; BCA determination of protein concentration test kit: green skies biotechnology research institute.
2. experimental technique
Trophophase cell of taking the logarithm is cultivated in 6 orifice plates, and adjustment cell concentration is 1 × 10
6individual/ml, experiment grouping: Normal group (NC); RhTNF-α damage model group (rhTNF-α) (l0ng/ml); Radix Arctii oligosaccharide three experimental grouies (62.5 μ g/ml, 125 μ g/ml, 250 μ g/ml).The Radix Arctii oligosaccharide effect 2h of each concentration is added in advance during experiment, add rhTNF-α (l0ng/ml) again and act on collecting cell culture fluid and cell sample after 4h, reference reagent box (nitrate reductase method) measures NO content in cell culture fluid and cell pyrolysis liquid respectively.
3. experimental result
Radix Arctii oligosaccharide hatches HY926 cell 2h in advance, then adds after rhTNF-α (l0ng/ml) stimulates 4h, in cell pyrolysis liquid and culture fluid shown in NO content following (see accompanying drawing 4).
A in Fig. 4: NO level in cell culture fluid; B: NO level in cell pyrolysis liquid; Compared with Normal group, ##P<0.01, ###P<0.001; Compared with model group, * P<0.05, * * P<0.01.
Experimental example 3: Radix Arctii oligosaccharide arterial thrombus is inhibited
1. Radix Arctii oligosaccharide is to the thrombotic inhibitory action of Mouse tail artery
1.1 experiment material
Bull Kun Ming mice 50, body weight 25g ± 2g, is divided into 5 groups at random, often organizes 10.Thered is provided (quality certification number: real dynamic No. 022nd, the word of the Liao Dynasty) by Dalian Medical Univ's SPF animal center.Carrageenin is provided by Sigma Co., USA.
1.2 experimental technique
Blank group: male Kunming white mice 10, not administration, not modeling, to isodose tri-distilled water.
Negative control group: male Kunming white mice 10, not administration, modeling, to isodose tri-distilled water.
Experimental agents group: male Kunming white mice 30, stochastic averagina is divided into three groups, gives the medicine of three kinds of variable concentrations respectively, modeling.
The low middle high dose group (2.5mg/10g, 5mg/10g, 10mg/10g) of Radix Arctii oligosaccharide.
Blank group and negative control group mice, with tri-distilled water gavage 7 days, have filled with half an hour after water on the 4th day, the normal saline of blank group subcutaneous injection 0.1ml/10g dosage, negative control group subcutaneous injection 0.1ml/10g dosage 0.4% carrageenin.Three Experimental agents group mices to the medicine 7 days of various dose, after the 4th day administration half an hour subcutaneous injection 0.1ml/10g dosage 0.4% carrageenin.Measure mouse tail length and thrombosis length after administration in 7th day, obtain thrombotic relative length RATL (%) according to thrombosis length and afterbody total length.
1.3 experimental result
Experiment proves, Radix Arctii inulin has significant inhibitory action to Mouse tail artery thrombosis, along with the increase inhibitory action of dosage increases (p<0.05), but the Experimental agents group of 2.5mg/10g dosage has no obvious effect (see accompanying drawing 5).
NS in Fig. 5: model control group; NBP1,2,3: be respectively Radix Arctii polysaccharide 2.5,5,10mg/10g group.Compare with NS group: * P<0.05.
2. Radix Arctii oligosaccharide is to the thrombotic inhibitory action of rat carotid artery
2.1 experiment material
FeCl
3(analytical pure): Tianjin great Mao chemical reagent factory; Heparin sodium (tiring as 150U/mg): Chinese Hui Shi biochemical reagents company limited; Institute of biological products is built up in adenosine diphosphate (ADP) disodium (ADP) Nanjing; BCA determination of protein concentration test kit: the green skies, Jiangsu biotechnology research institute; AT-III ELISA detection kit: east, Beijing scientific and technological biological company limited of song.
Healthy Sprague-Dawley(SD) rat, male and female half and half, body weight 180---220g, cleaning grade, is provided by Dalian Medical Univ's Experimental Animal Center, the quality certification number: SCXK(the Liao Dynasty) 2013-0003, room temperature, freely ingests and drinking-water.
2.2 experimental technique
60 healthy SD rats, be divided into following 6 groups at random: sham operated rats (Sham), model group (Model), Radix Arctii oligosaccharide high (8.8mg/kg), in (4.4mg/kg), low (2.2mg/kg) dosed administration group, heparin (I00U/kg) positive drug control group.After lumbar injection 20% urethane (50mg/kg) anesthesia, 20min left femoral vein injectable drug or equivalent blank solvent before modeling, common carotid artery thrombus model is made with reference to the improvement of Kurz method, get neck median incision, expose left common carotid artery 1cm long, its underlying small pieces plastic sheeting, common carotid artery is placed a suction 10 μ l25%FeCI.The little filter paper (5mm × 5mm) of solution.Remove filter paper after effect 5min, the scraps of paper remove rear l0min from abdominal aortic blood, and whole blood adds 3.8% sodium citrate anticoagulant by 9:1, and the centrifugal 10min of 1000rpm/min, gets supernatant, is platelet rich plasma (PlateletRichPlasma, PRP); Residual blood continues the centrifugal 10min of 3000rpm/min, gets supernatant, obtains platelet poor plasma (PoorPlateletPlasma, PPP).Return to zero with PPP, using PRP as platelet donation, 200 μ lPRP got by each sample, after 37 DEG C of incubation 5min, adding derivant ADP(final concentration is 1 μM), adopt turbidimetry to carry out Platelet mensuration, utilize LBY-NJ2 type four-way platelet aggregation instrument to trace curve of platelet aggregation, maximum platelet aggregation rate in record 5min.With reference to AT-III content in enzyme-linked immunosorbent assay kit measurement rat plasma.
2.3 experimental result
This research is first at FeCI
3the rat carotid artery thrombus model of induction observes Radix Arctii oligosaccharide there is certain anti-thrombosis function (see accompanying drawing 6), compared with Normal group, model group obviously forms thrombosis (P<0.0001) compared with model group, Radix Arctii oligosaccharide group has obvious inhibition thrombosis effect (low high dose group, P<0.05; Middle dosage group P<0.01).
By platelet aggregation test, calculate the display of maximum platelet aggregation rate (see accompanying drawing 7) result, compared with Normal group, ###P<0.0001; Compared with model group, each dosage group of Radix Arctii oligosaccharide all obviously can reduce platelet aggregation rate (low dose group, P<0.05; Middle high dose group P<0.01), reduce thrombotic risk.
Enzyme linked immunological experimental result display (accompanying drawing 8), compared with Normal group, ###P<0.0001; Compared with model group, AT-III concentration (low high dose group, P<0.05 in Radix Arctii oligosaccharide energy elevate plasma; Middle dosage group P<0.01), prompting Radix Arctii oligosaccharide can play blood coagulation resisting function by the effect strengthening AT-III, shows that Radix Arctii oligosaccharide anti thrombotic action may be relevant with enhancing anticoagulation system activity.
Radix Arctii oligosaccharide can be prepared into the oral formulations such as tablet, capsule by existing conventional method, also can be prepared into the injecting drug uses such as injection.
Claims (3)
1. the application in antithrombotic reagent prepared by a Radix Arctii oligosaccharide.
2. the application in antithrombotic reagent prepared by Radix Arctii oligosaccharide according to claim 1, it is characterized in that preparing the application in anticoagulant medicine.
3. the application in antithrombotic reagent prepared by Radix Arctii oligosaccharide according to claim 1, it is characterized in that the application in preparation protection vascular endothelial cell medicine.
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| CN111184729A (en) * | 2020-02-18 | 2020-05-22 | 博德生物技术（德州）有限公司 | Application of burdock inulin in preparation of medicine for treating hyperlipidemia |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111184729A (en) * | 2020-02-18 | 2020-05-22 | 博德生物技术（德州）有限公司 | Application of burdock inulin in preparation of medicine for treating hyperlipidemia |
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