CN103638034A - Application of morindae officinalis oligosaccharide pentasaccharide to preparation of drug for treating myocardial ischemia and reperfusion injury - Google Patents
Application of morindae officinalis oligosaccharide pentasaccharide to preparation of drug for treating myocardial ischemia and reperfusion injury Download PDFInfo
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Abstract
The invention discloses an application of a morindae officinalis extract and morindae officinalis oligosaccharide pentasaccharide to preparation of a drug for treating myocardial ischemia and/or reperfusion injury and promoting therapeutic angiogenesis. The myocardial ischemia and reperfusion injury belong to secondary injury brought to an organism when the hemoperfusion of myocardium is stopped or poor, i.e., after the myocardium is subjected to ischemia, hypoxia injury, circulation reinstitution and blood supply recovery. The morindae officinalis extract and the morindae officinalis oligosaccharide pentasaccharide both play a role in protecting an in-vivo rat myocardial ischemia reperfusion injury model and an in-vitro purification cultured neonatal rat myocardial cell hypoxia/reoxygenation injury model, and can be used for remarkably reducing the myocardial infarction area and the incidence rate of reperfusion arrhythmias, effectively protecting the form of a myocardial cell and relieving the injury of hypoxia/reoxygenation to the myocardial cell. The therapeutic angiogenesis promotion means that the aims of recovering the blood supply of ischemic myocardium and improving the heart function are achieved through increasing the functional coronary artery branch or side branch under the irritation actions of some methods, including the therapeutic actions of some drugs.
Description
Technical field
The present invention relates to the medicinal application field of plant extract, be specifically related to Radix Morindae Officinalis oligosaccharide preparation resist myocardial ischemia and reperfusion injury medicine in application.
Background technology
Coronary atherosclerotic heart disease (coronary heart disease, CAD), is called for short coronary heart disease, also referred to as ischemic heart desease, is the common cardiovascular disease of the mankind, is topmost adult's lethal cardiovascular disease.The acute myocardial infarction that coronary heart disease causes is to cause at present the main cause that cardiovascular disease incidence rate is high, mortality rate is high.Incidence of coronary heart disease continues to increase, and rejuvenation trend is obvious.Coronary heart disease shows serious myocardial ischemia disease, serious threat human health clinically.The mortality rate of patients with coronary heart disease is relevant to the left ventricular function that myocardial damage area determines with long-term prognosis, and myocardial damage area is by the degree of myocardial ischemia and Time dependent.Cardiac muscle, more than blood supply blocking-up 30min, will cause the Ultrastructural change of each organelle and dysfunction, causes the irreversible damage of myocardial cell even dead.Myocardial ischemic injury increases the weight of along with the prolongation of Ischemia Time.
Thrombolytic, arteria coronaria artery bypass and arteria coronaria intervention etc. are the effective ways for the treatment of acute myocardial infarction, it is logical again that these methods can make to block arteria coronaria, but pouring into again in time of myocardial ischemia, although can save part damage but not yet downright bad myocardial cell also may increase the weight of the damage of ischemic myocardium, the necrosis that shows as myocardial cell is accelerated, cellular swelling, the formation of muscle fibers contract band, myocardium internal hemorrhage and without perfusion phenomenon, also may produce serious reperfusion arrhythmia.The myocardial ischemia reperfusion injury (myocardial ischemical reperfusion injury, MIRI) occurring in the treatment of this coronary heart attack has had a strong impact on survival rate instant after coronary blood transport reconstruction and the recovery of Long-term Cardiac Function.
Radix Morindae Officinalis (Radix morinda officinalis, RMO), be Maguireothamnus speciosus (Rubiaceae), be one of four famous great Nan medicines of China, be also the rare medicinal herbs of foreign exchange earning.Radix Morindae Officinalis has kidney invigorating and YANG supporting, bone and muscle strengthening, and the effect of wind-damp dispelling, it is a kind of safe drugs for toxicological experiment proof.Have bibliographical information, the content of 5～7 years raw Radix Morindae Officinalis total sugar can surpass 50%, is the important component part of Radix Morindae Officinalis chemical composition.Activity research demonstration, Radix Morindae Officinalis carbohydrate content has immunomodulating, antitumor, antidepressant, anti-stress, antiinflammatory, antioxidation, the anti-ageing effect of waiting for a long time.
Summary of the invention
The present invention is particularly extracted from the dry root of Maguireothamnus speciosus Radix Morindae Officinalis (the Morinda officinalis How) biological activity of Radix Morindae Officinalis oligosaccharide (Morindae officinalis oligosaccharides, MOO) to Radix Morindae Officinalis extract is studied.Applicant finds unexpectedly, and Radix Morindae Officinalis extract is resisting myocardial ischemia and/or Reperfusion injury damaging the spleen and stomach has obvious activity.Its active component is followed the trail of and found that the effective ingredient of its Ischemic myocardium and reperfusion injury, the effect of promotion angiogenesis is the mixture of inulin type oligosaccharide; wherein in Radix Morindae Officinalis oligosaccharide monomer, 4,5,6 polysaccharide are to promoting Human umbilical vein endothelial cells (HUVEC) propagation and tubule formation effect after anoxia/reoxygenation the most obvious, and between three, effect is mutually collaborative.
Radix Morindae Officinalis oligosaccharide 4 polysaccharide molecular formula: C
24h
42o
21, molecular weight: 666.58, CAS accession number: 13133-07-8, chemical name: C24H42 O21 Nystose, structural formula I; Abbreviation Hex4.
Radix Morindae Officinalis oligosaccharide 5 polysaccharide molecular formula: C
30h
52o
26, molecular weight: 828.72, CAS accession number: 59432-60-9, chemical name: Fructofuranosyl nystose 1F-fructofuranosylnystose, structural formula II; Abbreviation Hex5.
Radix Morindae Officinalis oligosaccharide 6 polysaccharide molecular formula: C
36h
62o
31, molecular weight: 990.86, chemical name: sugarcane fruit six sugared Fru β (2-1)-[Fru β (2-1)] 4-α (2-1) Glc, structural formula II I, abbreviation Hex6.
Our research is found first; Radix Morindae Officinalis 4,5,6 polysaccharide are capable of reducing myocardial infarction area significantly; alleviate the damage of hypoxia-reoxygenation to myocardial cell; reduce again the ARR generation of flush phase; the form of effective protecting myocardial cell; promote Therapeutic Angiogenesis, its therapeutic effect is better than DIAOXINXUE KANG and Heart pill of Musk.Above-mentioned myocardial ischemia and reperfusion injury be cardiac muscle when hemoperfusion stops or is bad, experience ischemia, anoxia-induced apoptosis, and re-establish circulation, recover the rear damage again bringing to body of blood confession.Promote that Therapeutic Angiogenesis is that stimulation by certain methods comprises that the therapeutical effect of some drugs increases functional coronary arterial tree or side is propped up, reach and recover the confession of ischemic myocardium blood, improve the object of cardiac function.
Based on above-mentioned discovery, the object of the present invention is to provide Radix Morindae Officinalis extract particularly Radix Morindae Officinalis 4,5,6 polysaccharide in the new purposes of preparing aspect New Effective Drugs Against Myocardial Ischemia-Reperfusion Injury.
The invention provides that Radix Morindae Officinalis extract resists myocardial ischemia in preparation, reperfusion injury or promote the application in Therapeutic Angiogenesis medicine.
Further, described Radix Morindae Officinalis extract is extracted from the dry root of Maguireothamnus speciosus Radix Morindae Officinalis.
Further, the invention provides that Radix Morindae Officinalis oligosaccharide resists myocardial ischemia in preparation, reperfusion injury or promote the application in Therapeutic Angiogenesis medicine.Preferably, described Radix Morindae Officinalis oligosaccharide comprises one or more of compound shown in structural formula I, structural formula II and structural formula II I
Further, the invention provides a kind of pharmaceutical composition, it is characterized in that, in described pharmaceutical composition, comprise Radix Morindae Officinalis oligosaccharide component.Preferably, described Radix Morindae Officinalis oligosaccharide comprises one or more of compound shown in said structure formula I, structural formula II and structural formula II I.Pharmaceutical composition, its dosage form can be tablet, pill or capsule, injection etc.
Further, the invention provides a kind of preparation method of Radix Morindae Officinalis oligosaccharide 4,5,6 polysaccharide, comprise the steps:
1), Radix Morindae Officinalis extract is dissolved in the water, then lyophilization;
2), in freeze-dried thing, add pyridine, and stir below at 0 ℃, limit adds Glacial acetic acid;
3), under chamber state, stir after 24h concentrating under reduced pressure;
4), residual fraction adds CHCl
3, with saturated soda water, saturated aqueous common salt, clean;
5), with after dried over mgso, then CHCl is removed in decompression
3;
6), with compound method for refining, purify respectively, expansion solvent is toluene: ethyl acetate=2:1 → 1:1 → 1:2 → 1:3 → 1:5, obtains respectively acetylation Radix Morindae Officinalis oligosaccharide 4,5,6 polysaccharide monomers;
7), magnetic resonance detection is determined respectively resulting acetylation Radix Morindae Officinalis oligosaccharide 4,5,6 polysaccharide monomer structures;
8), acetylation Radix Morindae Officinalis oligosaccharide 4,5,6 polysaccharide monomers are deacetylated;
In the MeOH that acetylation Radix Morindae Officinalis oligosaccharide 4,5,6 polysaccharide monomers are dissolved in, then the CH adding
3oNa, at room temperature stirs after 2.5h, then adds Dowex50W x2[H+] neutralize, after resin filter, solvent is removed in decompression, and residual fraction is dissolved into H
2in O, by Bio-Gel P-2Gel:10mL, carry out desalination, freeze-dried after, obtain white solid;
9), white solid obtains Radix Morindae Officinalis oligosaccharide 4 polysaccharide sterlings after the gel filtration of MALDI-TOF-MS
Wherein, described myocardial ischemia reperfusion injury, for cardiac muscle is when hemoperfusion stops or be bad, experiences after ischemia, anoxia, and circulation re-establishes, and blood is for the damage bringing to body after recovering.
Radix Morindae Officinalis 4,5,6 polysaccharide of the present invention are extracted from the dry root of Maguireothamnus speciosus Radix Morindae Officinalis (Morinda officinalis How).
Medicine of the present invention refers to that take Radix Morindae Officinalis 4,5,6 polysaccharide is main active, and acceptable preparation in the medical treatment made from pharmaceutically suitable carrier, as tablet, pill or capsule etc.
Medicine of the present invention, its dose is in Radix Morindae Officinalis 4,5,6 polysaccharide: 0.5g/ time or 1.5g/ days.
Radix Morindae Officinalis 4,5,6 polysaccharide of the present invention can, with directly obtaining by commercially available, also can obtain by plant extract.
The present invention passes through: 1. isolated cells experimental system: neonatal rat myocyte anoxia/reoxygenation, apoptosis, and after Human umbilical vein endothelial cells (HUVEC) anoxia/reoxygenation, propagation, migration and tubule form; 2. Integral animal experiment system: non-invasive, have wound property myocardial ischemia/Infarction Model; Radix Morindae Officinalis 4,5,6 researchs of polysaccharide to cardiovascular protection effect have been carried out.
Beneficial effect of the present invention is:
1, the invention provides and take 4,5,6 polysaccharide as the main new purposes of Radix Morindae Officinalis oligosaccharide, it has good protective effect to cardiovascular system.
Consulting on the basis of a large amount of pertinent literatures both at home and abroad, effect to Radix Morindae Officinalis 4,5,6 polysaccharide protection cardiovascular systeies is studied, result confirms: 1) the anoxia reoxygenation injury of neonatal cardiac myocytes model of rat in vivo Model of Myocardial Ischemia-Reperfusion Injury and in vitro purification cultivation is all had to protective effect, significantly capable of reducing myocardial infarction area, alleviate again the ARR generation of flush phase, effectively the form of protecting myocardial cell, alleviates the damage of hypoxia-reoxygenation to myocardial cell; 2) can improve rat myocardial cell antioxidase (SOD, LDH, GSH-Px) level, lipid peroxidation injury degree while alleviating rat myocardial cell ischemical reperfusion injury, reduces the generation of lipid peroxidation product MDA; 3) iNOS expression and NO output in the time of can improving rat myocardial ischemia and reperfusion, improve myocardial cell Ca
2+-ATPase and Na
+-K
+-ATPase is active; 4), by the related gene expression of regulating cell apoptosis, can significantly alleviate the apoptosis of myocardial cell; 5) secretion of ET-1 content and TNF-α, IL-1 β, IL-6 during by reduction cardiomyocyte injury induced by hypoxia/reoxygenation, the inflammatory reaction degree while significantly improving Myocytes Anoxia reoxygenation injury; 6) can improve the hemodynamics of blood stasis rat, anticoagulant; 7) Radix Morindae Officinalis sugar chain can significantly promote sarcoplast propagation and Cardiocytes differentiation, be enhanced to the PCNA positive reaction of myocyte's karyon, improve the expression of TGF-β 1, TGF-β 2, GATA-4 gene and albumen in sarcoplast, raise the expression of information conduct factors Smad4, according to the expression of dosage two-ways regulation Idiotype information conduct factors p-Smad2/3 and Smad7.8) can effectively promote angiogenesis, significantly alleviate the damage of H/R to vascular endothelial cell, inhibited apoptosis, effectively promote endothelial cell migration and the vascularization of H/R damage, improve ischemic myocardium microvessel density, the significantly expression of vegf protein, bFGF albumen, Flt-1 albumen, TRPC6 albumen, ERK1/2 albumen and PDGF-B albumen in induction of vascular Newborn Process.The main signal molecule proteins such as regulation and control Notch1/Jagged1 are expressed, thereby reduce the expression of Notch signal and pten protein.9) can activate large conductance calcium-activated potassium (BKCa) passage on endothelial cellular membrane.10) Radix Morindae Officinalis sugar chain has doses dependency to the protective effect of cardiovascular system.
2, Radix Morindae Officinalis 4,5,6 polysaccharide that the present invention adopts are extracted from pure natural raw material, and extracting method is simple, and chemical constitution and molecular weight are clear and definite, stable in properties, and extraction ratio is high, quality controllable.
Accompanying drawing explanation
Fig. 1 is myocardial cell morphological observation (* 40), and A is normal myocardium cell, and B is H/R group, and C is H/R+ DIAOXINXUE KANG group, and D is H/R+4 polysaccharide group, and E is H/R+5 polysaccharide group, and F is H/R+6 polysaccharide group;
Fig. 2 is that Myocardial ultrastructure is observed (* 5000), and A is normal myocardium cell, and B is H/R group, and C is H/R+ DIAOXINXUE KANG group, and D is H/R+4 polysaccharide group, and E is H/R+5 polysaccharide group, and F is H/R+6 polysaccharide group;
Fig. 3 is myocardial infarction tissue specimen substantially, and A is normal myocardium, and B is MI group, and C is MI+ DIAOXINXUE KANG group, and D is MI+4 polysaccharide group, and E is MI+5 polysaccharide group, and F is MI+6 polysaccharide group;
Fig. 4 is the detection of ischemic myocardium microvessel density (MVD), and A is normal myocardium, and B is MI group, and C is MI+ Heart pill of Musk group, and D is MI+4 polysaccharide group, and E is MI+5 polysaccharide group, and F is MI+6 polysaccharide group;
Fig. 5 is propagation (* 10) after HUVEC anoxia/reoxygenation (H/R), and A is normal group, and B is H/R group, and C is H/R+ Heart pill of Musk group, and D is H/R+4 polysaccharide group, and E is H/R+5 polysaccharide group, and F is H/R+6 polysaccharide group;
Fig. 6 HUVEC moves (* 10), and A is normal group, and B is H/R group, and C is H/R+ Heart pill of Musk group, and D is H/R+4 polysaccharide group, and E is H/R+5 polysaccharide group, and F is H/R+6 polysaccharide group;
Fig. 7 HUVEC tubule forms (* 10), and A is normal group, and B is H/R group, and C is H/R+ Heart pill of Musk group, and D is H/R+4 polysaccharide group, and E is H/R+5 polysaccharide group, and F is H/R+6 polysaccharide group;
Fig. 8 HUVEC tubule forms (* 10), and A is normal group, and B is H/R group, and C is H/R+ Heart pill of Musk group, and D is H/R+MOO group, and E is H/R+Hex4, and F is H/R+Hex5, and G is H/R+Hex6;
The specific embodiment
The experiment materials such as biochemical reagents, laboratory animal and cell used in the present invention all can obtain by commercially available channel.Radix Morindae Officinalis medical material (Radix morinda officinalis), purchased from Deqing County, Guangdong Province medical material company, is accredited as Grade A through College of Pharmacy, Henan College of Traditional Chinese Medicine; Radix Morindae Officinalis oligosaccharide mixture (Morinda officinalis oligosaccharides) adopts this area conventional method prepare and determine; SD/Wistar rat is purchased from Zhengzhou University's Experimental Animal Center; Human umbilical vein endothelial cells (HUVEC) is purchased from Wuhan University's cell preservation center, and above-mentioned material also can be bought and be obtained by other commercial sources.
Embodiment 1
The preparation of Radix Morindae Officinalis oligosaccharide 4,5,6 polysaccharide monomers:
(1) 10g Radix Morindae Officinalis extract is dissolved in to the H of 80ml
2in O, then make it freeze-dried.
(2) in the freeze-dried thing of 8.3g, add the pyridine of 100ml, and stir below the temperature of 0 ℃, limit adds the Glacial acetic acid of 100ml.
(3) under chamber state, stir after 24h, by decompression method, remove unnecessary medium.
(4) residual fraction adds CHCl again
3, with saturated soda water, saturated aqueous common salt, clean.
(5) with after dried over mgso, then CHCl is removed in decompression
3.
(6) with compound method for refining, purify respectively and (launch solvent: toluene: ethyl acetate (Toluene:Ethyl acetate)=2:1 → 1: 1 → 1:2 → 1: 3 → 1:5).Extract the acetylation Hex4 monomer (Hex4 perAc) of 623mg.Extract the acetylation Hex5 monomer (Hex5 perAc) of 623mg.Extract the acetylation Hex6 monomer (Hex6 perAc) of 74mg.
(7) with magnetic nuclear resonance method, determine Hex4 perAc, Hex5 perAc, Hex6 perAc structure.
(8) Hex4 perAc, Hex5 perAc, Hex6 perAc are deacetylated:
The Hex4 perAc of 100mg is dissolved in the MeOH of 5mL, then adds 18mg, the CH of 0.3eq
3ona.Under greenhouse, stir after 2.5h, then add Dowex50W x2[H+] neutralize.After resin filter, solvent is removed in decompression.Residual fraction is dissolved into H
2in O, by (Bio-Gel P-2Gel:10mL), carry out desalination.After freeze-dried, can obtain white solid (57mg).
The Hex5 perAc of 100mg is dissolved in the MeOH of 5mL, then adds 18mg, the CH of 0.3eq
3ona.Under greenhouse, stir after 2.5h, then add Dowex50W x2[H+] neutralize.After resin filter, solvent is removed in decompression.Residual fraction is dissolved into H
2in O, by (Bio-Gel P-2Gel:10mL), carry out desalination.After freeze-dried, can obtain white solid (57mg).
The Hex6 perAc of 74mg is dissolved in the MeOH of 5mL, then adds 12mg, the CH of 0.3eq
3ona.Under greenhouse, stir after 2.5h, then add Dowex50W x2[H+] neutralize.After resin filter, solvent is removed in decompression.Residual fraction is dissolved into H
2in O, by (Bio-Gel P-2Gel:10mL), carry out desalination.After freeze-dried, can obtain white solid (39mg).
(9) above-mentioned white solid is carried out respectively after the gel filtration of MALDI-TOF-MS, obtain Hex4,5,6 sterlings.
Hex4: purity more than 95% (NMR); Preserve 4 ℃; Molecular weight 666.578;
Hex5: purity more than 95% (NMR); Preserve 4 ℃; Molecular weight 828.7183;
Hex6: purity more than 95% (NMR); Preserve 4 ℃; Molecular weight 990.859.
Embodiment 2
(1) in vitro endotheliocyte and Integral animal experiment
<1> is in the experimental system of Neonatal Mouse myocardial cell, adopt newborn (1-3d) SD/Wistar rat, the dirty primary myocardial cell culture of doing of coring, be divided into Normal group, Hypoxia/Reoxygenation Injury (H/R) model group, H/R+ positive drug (DIAOXINXUE KANG, 0.7gL
-1) matched group, H/R+Hex4 (42nmolml
-1), Hex5 (30nmolml
-1), Hex6 (22nmolml
-1) each group.Take myocardial cell form and Myocardial ultrastructure as main evaluation index, observe Radix Morindae Officinalis 4,5,6 protective effects of polysaccharide to myocardial cell.
<2>, in the experimental system of Human umbilical vein endothelial cells (HUVEC) model of angiogenesis, is divided into Normal group, H/R model group, H/R+ positive drug (Heart pill of Musk, 2gL
-1) matched group, H/R+Hex4 (42nmolm1
-1), Hex5 (30nmolml
-1), Hex6 (22nmolml
-1) each group.Take vascular endothelial cell proliferation and endotheliocyte is arranged in luminal structure as main evaluation index, the anoxia model group cell proliferation of usining become with cell arrangement luminal structure number higher than two standard deviations of normal control class mean as the successful evaluation criterion of model.After model success, in 12h, give respectively and different medicines.
After administration, 1. 6h, 12h, 24h, 48h, 72h measure by fluidic cell method the propagation of respectively organizing vascular endothelial cell respectively; 2. with flow cytometer methods analyst, respectively organize cell generation cycle.3. adopt transwell cell migration experiment and tubule form the propagation of the short endotheliocyte of laboratory observation Radix Morindae Officinalis 4,5,6 polysaccharide and induce it to be differentiated to form the effect of tube chamber.
(2) Integral animal experiment
The non-invasive myocardial infarction and ischemia model of <1>: select SD/Wistar rat, animal is divided at random: blank (normal saline) matched group, isoproterenol (Isoproterenol) Chronic myocardium ischemia damage model group, model+positive drug (DIAOXINXUE KANG, 28mgkg
-1d
-1) matched group, model+Hex4 (24mgkg
-1d
-1), Hex5 (12mgkg
-1d
-1), Hex6 (2.8mgkg
-1d
-1) each group.Except blank group intraperitoneal injection of saline, all the other each groups all adopt repeatedly isoproterenol lumbar injection Methods of Preparing Myocardial chronic ischemia damage model, take the electrocardiogram ST-T changes as decision model success foundation.Each group all after model success, in 24h, start to pharmaceutical intervention (blank group, model group gavage to isometric normal saline, each administration group gavage is given the medicine with corresponding isometric(al) various dose).
<2> has wound property myocardial infarction model: select SD/Wistar rat, be divided at random false ligation group, Infarction Model group, model+positive drug (Heart pill of Musk group 30mgkgd) matched group, model+Hex4 (24mgkg
-1d
-1), Hex5 (12mgkg
-1d
-1), Hex6 (2.8mgkg
-1d
-1) each group.After false ligation group is opened the separated coronary artery of breast a not ligation of threading, all the other each groups are all in the slightly lower 1～2mm of pulmonary conus and left auricle boundary place following coronary artery occlusion left anterior descending branch, prepare myocardial infarction model, the cardiac muscle under ligation position of take bleaches, beat and weaken and electrocardiogram occurs that the ST section back of a bow is upwards obviously raised as modeling and successfully indicates.Each group all after modeling, in 24h, start pharmaceutical intervention (false ligation group, Infarction Model group lumbar injection to isometric normal saline, each administration group lumbar injection is given and corresponding isometric(al) various dose medicine).
After pharmaceutical intervention 6w, put to death animal, cardiac muscle is drawn materials: 1. specimens paraffin embedding slices, the general Histomorphological of row; 2. HE staining examine is respectively organized microvessel density in ischemic myocardium in rat (MVD), from integral level, observes the feature that Radix Morindae Officinalis 4,5,6 polysaccharide promote angiogenesis.
Experimental result is as shown in the figure:
Fig. 1 is the impact of Radix Morindae Officinalis 4,5,6 on neonatal rat myocardial cell form: under inverted microscope, can see that normal myocardium cell culture is after 3 days, the growth of cell cluster, stretches out pseudopodium, and refractivity is strong, and beats obviously.Cellular morphology presents variation, as circle, fusiformis and vertebra shape (figure A).After anoxia/reoxygenation, myocardial cell pseudopodium shortens or disappears, and refractivity declines, and beats and weakens or disappear (figure B).DIAOXINXUE KANG group myocardial cell form is compared with anoxia/reoxygenation group have some improvement (figure C).Hex4, Hex5, each administration group myocardial cell form of Hex6 be improved significantly (figure D, E, F).
Fig. 2 is Radix Morindae Officinalis 4,5,6 on the Ultrastructural impact of neonatal rat myocardial cell: under transmission electron microscope, Myocardial ultrastructure shows, normal cell cultivation group ultrastructure is complete, clear, and organelle is many, nuclear membrane smooth surface, in core, chromatin is loose, density homogeneous (figure A).The visible cell space of Hypoxia-reoxygenation model group diminishes, nuclear membrane shrinkage, chromatic agglutination, have inhomogeneous limit collection phenomenon, part chromatin gathers and is the change of demilune sample, nuclear hyperchromatism under complete nuclear membrane, pyknosis, mitochondrion has the apoptosis representative configuration features such as vacuolar degeneration (figure B).DIAOXINXUE KANG group cell ultrastructure obtains improvement in various degree compared with anoxia/reoxygenation group, but still has the slight limit of chromatin collection, the morphological change such as nuclear hyperchromatism (figure C).Hex4, Hex5, each administration group cell ultrastructure of Hex6 approach normal (figure D, E, F).
Fig. 3 is the impact of Radix Morindae Officinalis 4,5,6 on rat myocardial infarction model: visible in myocardial infarction cardinal principle tissue specimen, to compare with normal group (figure A), and myocardial infarction model group cardiac muscle is the saturating wall ischemia of large area downright bad (figure B).DIAOXINXUE KANG group myocardial ischemia necrosis area alleviates (figure C) to some extent compared with model group.Hex4, Hex5, each administration group myocardial ischemia necrosis area of Hex6 and degree all obviously alleviate (figure D, E, F) compared with model group.
Fig. 4 is the impact of Radix Morindae Officinalis 4,5,6 on rat heart muscle blood vessel hyperplasia and microvessel density: under light microscopic, in visible cardiac muscle section, false ligation group, model group, Heart pill of Musk group, Hex4, Hex5, each administration group of Hex6 all can be seen newborn blood capillary; But not obvious (the figure A of the visible blood capillary proliferation of false ligation group.The visible a small amount of blood capillary proliferation of model group (figure B).Heart pill of Musk group and Hex4, Hex5, Hex6 respectively organize the blood capillary (figure C, D, E, F) of visible most hypertrophy.Compare with false ligation group, model group rat MVD (microvessel density) significantly increases (P < 0.05).Compare with model group, Heart pill of Musk group and Hex4, each group of Hex5, Hex6 all can significantly increase MVD (P < 0.05).
Fig. 5 is the impact of Radix Morindae Officinalis 4,5,6 on cell proliferation of human umbilical vein: with normal group comparison, and anoxia/reoxygenation group HUVEC cell proliferation quantity less (figure B).Compare with model group, Heart pill of Musk group cell proliferation quantity increases, and statistics has significant difference (P < 0.05, figure C).Compare with Heart pill of Musk group, Hex4, Hex5, each medication group of Hex6 all can significantly promote the propagation (P < 0.05, figure D, E, F) after cell injury.
Fig. 6 is affect 1 (the cell method) of Radix Morindae Officinalis 4,5,6 on Human umbilical vein endothelial cells migration: compare with normal group (figure A), anoxia/reoxygenation (H/R) model group cell migration number obviously reduces (P<0.05, figure B).Positive control drug Heart pill of Musk group does not show the effect (figure C) that promotes anoxia/reoxygenation endothelial cell migration.Hex4, Hex5, each group of Hex6 can obviously increase the migration number (P<0.05, figure, D, E, F) of cell after anoxia/reoxygenation.
Fig. 7 is affect 2 (scarifications) of Radix Morindae Officinalis 4,5,6 on Human umbilical vein endothelial cells migration: compare with normal group (figure A), anoxia/reoxygenation (H/R) model group cell migration number obviously reduces (P<0.05, figure B).Positive control drug Heart pill of Musk group has the trend (figure C) that promotes anoxia/reoxygenation endothelial cell migration.Hex4, Hex5, each group of Hex6 can obviously increase the migration number (P<0.05, figure D, E, F) of cell after anoxia/reoxygenation.
Fig. 8 is the impact that the short Human umbilical vein endothelial cells tubule of Radix Morindae Officinalis 4,5,6 forms: compare with normal group (figure A), MOO, Hex4, Hex5, Hex6 respectively organize cell culture 6h the intercellular form that interconnects as seen, 18h forms complete tube chamber spline structure on martrigel surface, and along with the prolongation of incubation time, luminal structure increases (figure D, E, F, G).Anoxia/reoxygenation model group cell just starts to interconnect after 18h, and 24h forms complete tube chamber spline structure and is less than MOO, Hex4, each group of Hex5, Hex6 (figure B) on matrigel surface.The tubule formation situation of positive control drug Heart pill of Musk group is worse than MOO, Hex4, each group of Hex5, Hex6 (figure C).Machine image analysis software is processed and is obtained the demonstration of tubule formation area as calculated, model group is compared significant difference (P<0.05) with normal group, MOO, Hex4, each group of Hex5, Hex6 are compared significant difference (P<0.05) with model group, model group is compared zero difference significantly (P>0.05) with Heart pill of Musk group.
More than describe preferred embodiment of the present invention in detail.The ordinary skill that should be appreciated that this area just can design according to the present invention be made many modifications and variations without creative work.Therefore, all technical staff in the art, all should be in the determined protection domain by claims under this invention's idea on the basis of existing technology by the available technical scheme of logical analysis, reasoning, or a limited experiment.
Claims (5)
- Radix Morindae Officinalis oligosaccharide 5 polysaccharide in preparation, resist myocardial ischemia, reperfusion injury or promote the application in Therapeutic Angiogenesis medicine.
- 2. application as claimed in claim 1, wherein, described Radix Morindae Officinalis oligosaccharide 5 glycan structures are as shown in structural formula II
- 3. a pharmaceutical composition, is characterized in that, comprises the 5 polysaccharide components of the Radix Morindae Officinalis oligosaccharide shown in structural formula II in claim 2 in described pharmaceutical composition.
- 4. pharmaceutical composition as claimed in claim 3, its dosage form is injection, tablet, pill or capsule.
- 5. a preparation method for Radix Morindae Officinalis oligosaccharide 5 polysaccharide, comprises the steps:1), Radix Morindae Officinalis extract is dissolved in the water, then lyophilization;2), in freeze-dried thing, add pyridine, and stir below at 0 ℃, limit adds Glacial acetic acid;3), under chamber state, stir after 24h concentrating under reduced pressure;4), residual fraction adds CHCl 3, with saturated soda water, saturated aqueous common salt, clean;5), with after dried over mgso, then CHCl is removed in decompression 3;6), with compound method for refining, purify, expansion solvent is toluene: ethyl acetate=2:1 → 1:1 → 1:2 → 1:3 → 1:5, obtains acetylation Radix Morindae Officinalis oligosaccharide 5 polysaccharide monomers;7), magnetic resonance detection is determined the structure of acetylation Radix Morindae Officinalis oligosaccharide 5 polysaccharide monomers;8), acetylation Radix Morindae Officinalis oligosaccharide 5 polysaccharide monomers are deacetylated;In the MeOH that acetylation Radix Morindae Officinalis oligosaccharide 5 polysaccharide monomers are dissolved in, then the CH adding 3oNa, at room temperature stirs after 2.5h, then adds Dowex50W x2[H+] neutralize, after resin filter, solvent is removed in decompression, and residual fraction is dissolved into H 2in O, by Bio-Gel P-2Gel:10mL, carry out desalination, freeze-dried after, obtain white solid;9), white solid obtains Radix Morindae Officinalis oligosaccharide 5 polysaccharide sterlings after the gel filtration of MALDI-TOF-MS.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113440540A (en) * | 2021-08-03 | 2021-09-28 | 南京理工大学 | Application of galacto-glucan in preparation of medicine for treating and/or preventing ischemia-reperfusion injury |
| CN114366752A (en) * | 2021-12-30 | 2022-04-19 | 冯国清 | Therapeutic effect of Morinda officinalis oligosaccharide 5 glycan monomer Hex5 on osteosarcoma |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103638162A (en) * | 2013-11-29 | 2014-03-19 | 郑州大学 | Application of morindae officinalis oligosaccharide tetrasaccharide to preparation of drug for treating myocardial ischemia and reperfusion injury |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103638162A (en) * | 2013-11-29 | 2014-03-19 | 郑州大学 | Application of morindae officinalis oligosaccharide tetrasaccharide to preparation of drug for treating myocardial ischemia and reperfusion injury |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113440540A (en) * | 2021-08-03 | 2021-09-28 | 南京理工大学 | Application of galacto-glucan in preparation of medicine for treating and/or preventing ischemia-reperfusion injury |
| CN113440540B (en) * | 2021-08-03 | 2022-03-18 | 南京理工大学 | Application of galacto-glucan in preparation of medicine for treating and/or preventing ischemia-reperfusion injury |
| EP4129305A1 (en) * | 2021-08-03 | 2023-02-08 | Nanjing Southern Element Biotechnology Co., Ltd. | Use of oligosaccharide riclinoctaose in preparation of drug for treating and/or preventing ischemia-reperfusion injury (iri) |
| CN114366752A (en) * | 2021-12-30 | 2022-04-19 | 冯国清 | Therapeutic effect of Morinda officinalis oligosaccharide 5 glycan monomer Hex5 on osteosarcoma |
| CN114366752B (en) * | 2021-12-30 | 2023-09-29 | 冯国清 | Therapeutic effect of morinda-oligosaccharide 5-glycan monomer Hex5 on osteosarcoma |
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Application publication date: 20140319 |