CN115778985A - Preparation method and application of stiff silkworm extract - Google Patents
Preparation method and application of stiff silkworm extract Download PDFInfo
- Publication number
- CN115778985A CN115778985A CN202211469580.XA CN202211469580A CN115778985A CN 115778985 A CN115778985 A CN 115778985A CN 202211469580 A CN202211469580 A CN 202211469580A CN 115778985 A CN115778985 A CN 115778985A
- Authority
- CN
- China
- Prior art keywords
- extract
- dialysis
- group
- stiff silkworm
- bombyx batryticatus
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 239000000284 extract Substances 0.000 title claims abstract description 105
- 241000255789 Bombyx mori Species 0.000 title claims abstract description 79
- 238000002360 preparation method Methods 0.000 title claims abstract description 19
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 52
- 239000003814 drug Substances 0.000 claims abstract description 32
- 238000000502 dialysis Methods 0.000 claims abstract description 31
- 235000019441 ethanol Nutrition 0.000 claims abstract description 30
- 238000001556 precipitation Methods 0.000 claims abstract description 17
- 238000000605 extraction Methods 0.000 claims abstract description 16
- 208000026106 cerebrovascular disease Diseases 0.000 claims abstract description 13
- 238000010438 heat treatment Methods 0.000 claims abstract description 13
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims abstract description 11
- 201000010099 disease Diseases 0.000 claims abstract description 10
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 8
- 238000002791 soaking Methods 0.000 claims abstract description 7
- 238000001704 evaporation Methods 0.000 claims abstract description 6
- 230000001954 sterilising effect Effects 0.000 claims abstract description 3
- 241000255791 Bombyx Species 0.000 claims description 52
- 239000000243 solution Substances 0.000 claims description 39
- 229940079593 drug Drugs 0.000 claims description 17
- 208000032382 Ischaemic stroke Diseases 0.000 claims description 13
- 238000000034 method Methods 0.000 claims description 13
- 239000000203 mixture Substances 0.000 claims description 3
- 230000008020 evaporation Effects 0.000 claims description 2
- 239000000706 filtrate Substances 0.000 claims description 2
- 239000007927 intramuscular injection Substances 0.000 claims description 2
- 238000010255 intramuscular injection Methods 0.000 claims description 2
- 238000001990 intravenous administration Methods 0.000 claims description 2
- 239000000546 pharmaceutical excipient Substances 0.000 claims description 2
- 239000000825 pharmaceutical preparation Substances 0.000 claims description 2
- 238000012869 ethanol precipitation Methods 0.000 claims 2
- 239000004615 ingredient Substances 0.000 claims 1
- 230000000694 effects Effects 0.000 abstract description 20
- HTTJABKRGRZYRN-UHFFFAOYSA-N Heparin Chemical compound OC1C(NC(=O)C)C(O)OC(COS(O)(=O)=O)C1OC1C(OS(O)(=O)=O)C(O)C(OC2C(C(OS(O)(=O)=O)C(OC3C(C(O)C(O)C(O3)C(O)=O)OS(O)(=O)=O)C(CO)O2)NS(O)(=O)=O)C(C(O)=O)O1 HTTJABKRGRZYRN-UHFFFAOYSA-N 0.000 abstract description 9
- 229960002897 heparin Drugs 0.000 abstract description 9
- 229920000669 heparin Polymers 0.000 abstract description 9
- 238000012360 testing method Methods 0.000 abstract description 6
- 238000010923 batch production Methods 0.000 abstract description 2
- 241000700159 Rattus Species 0.000 description 58
- 210000002381 plasma Anatomy 0.000 description 47
- 210000004556 brain Anatomy 0.000 description 23
- 210000004027 cell Anatomy 0.000 description 18
- 102000000429 Factor XII Human genes 0.000 description 17
- 108010080865 Factor XII Proteins 0.000 description 17
- 230000002490 cerebral effect Effects 0.000 description 17
- 239000002207 metabolite Substances 0.000 description 14
- 210000005013 brain tissue Anatomy 0.000 description 13
- 230000014509 gene expression Effects 0.000 description 12
- 210000004369 blood Anatomy 0.000 description 9
- 239000008280 blood Substances 0.000 description 9
- 210000003710 cerebral cortex Anatomy 0.000 description 9
- 229940019334 heparin group antithrombotic drug Drugs 0.000 description 9
- 102000004169 proteins and genes Human genes 0.000 description 9
- 108090000623 proteins and genes Proteins 0.000 description 9
- 241001465754 Metazoa Species 0.000 description 8
- 230000001054 cortical effect Effects 0.000 description 8
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 7
- 102000005789 Vascular Endothelial Growth Factors Human genes 0.000 description 7
- 108010019530 Vascular Endothelial Growth Factors Proteins 0.000 description 7
- 238000001514 detection method Methods 0.000 description 7
- 210000004925 microvascular endothelial cell Anatomy 0.000 description 7
- 239000000047 product Substances 0.000 description 7
- 210000002966 serum Anatomy 0.000 description 7
- 208000019553 vascular disease Diseases 0.000 description 7
- 208000029812 Cerebral Small Vessel disease Diseases 0.000 description 6
- 230000010100 anticoagulation Effects 0.000 description 6
- 238000003556 assay Methods 0.000 description 6
- 208000028867 ischemia Diseases 0.000 description 6
- 102000009123 Fibrin Human genes 0.000 description 5
- 108010073385 Fibrin Proteins 0.000 description 5
- BWGVNKXGVNDBDI-UHFFFAOYSA-N Fibrin monomer Chemical compound CNC(=O)CNC(=O)CN BWGVNKXGVNDBDI-UHFFFAOYSA-N 0.000 description 5
- 206010061218 Inflammation Diseases 0.000 description 5
- 102100024616 Platelet endothelial cell adhesion molecule Human genes 0.000 description 5
- 229950003499 fibrin Drugs 0.000 description 5
- 230000004054 inflammatory process Effects 0.000 description 5
- 239000012188 paraffin wax Substances 0.000 description 5
- 230000010410 reperfusion Effects 0.000 description 5
- 206010002091 Anaesthesia Diseases 0.000 description 4
- 101000738771 Homo sapiens Receptor-type tyrosine-protein phosphatase C Proteins 0.000 description 4
- 102000003777 Interleukin-1 beta Human genes 0.000 description 4
- 108090000193 Interleukin-1 beta Proteins 0.000 description 4
- 102000003814 Interleukin-10 Human genes 0.000 description 4
- 108090000174 Interleukin-10 Proteins 0.000 description 4
- 102000004388 Interleukin-4 Human genes 0.000 description 4
- 108090000978 Interleukin-4 Proteins 0.000 description 4
- 102000004889 Interleukin-6 Human genes 0.000 description 4
- 108090001005 Interleukin-6 Proteins 0.000 description 4
- 102100037422 Receptor-type tyrosine-protein phosphatase C Human genes 0.000 description 4
- 230000003044 adaptive effect Effects 0.000 description 4
- 230000037005 anaesthesia Effects 0.000 description 4
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 4
- 230000003542 behavioural effect Effects 0.000 description 4
- 238000010241 blood sampling Methods 0.000 description 4
- 210000001168 carotid artery common Anatomy 0.000 description 4
- 238000006243 chemical reaction Methods 0.000 description 4
- 238000010166 immunofluorescence Methods 0.000 description 4
- 239000012528 membrane Substances 0.000 description 4
- 230000004112 neuroprotection Effects 0.000 description 4
- 229910052760 oxygen Inorganic materials 0.000 description 4
- 239000001301 oxygen Substances 0.000 description 4
- 102000017915 BDKRB2 Human genes 0.000 description 3
- 101150022344 BDKRB2 gene Proteins 0.000 description 3
- 101100066087 Brassica napus bkr2 gene Proteins 0.000 description 3
- 238000002965 ELISA Methods 0.000 description 3
- JOYRKODLDBILNP-UHFFFAOYSA-N Ethyl urethane Chemical compound CCOC(N)=O JOYRKODLDBILNP-UHFFFAOYSA-N 0.000 description 3
- 102000001399 Kallikrein Human genes 0.000 description 3
- 108060005987 Kallikrein Proteins 0.000 description 3
- 230000004913 activation Effects 0.000 description 3
- 230000002146 bilateral effect Effects 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 238000011049 filling Methods 0.000 description 3
- 238000003384 imaging method Methods 0.000 description 3
- RAXXELZNTBOGNW-UHFFFAOYSA-N imidazole Natural products C1=CNC=N1 RAXXELZNTBOGNW-UHFFFAOYSA-N 0.000 description 3
- 210000002865 immune cell Anatomy 0.000 description 3
- 230000008595 infiltration Effects 0.000 description 3
- 238000001764 infiltration Methods 0.000 description 3
- 239000003112 inhibitor Substances 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 238000011068 loading method Methods 0.000 description 3
- 230000001404 mediated effect Effects 0.000 description 3
- 239000002504 physiological saline solution Substances 0.000 description 3
- 239000013641 positive control Substances 0.000 description 3
- 238000004393 prognosis Methods 0.000 description 3
- 230000035755 proliferation Effects 0.000 description 3
- 210000001519 tissue Anatomy 0.000 description 3
- HRPVXLWXLXDGHG-UHFFFAOYSA-N Acrylamide Chemical compound NC(=O)C=C HRPVXLWXLXDGHG-UHFFFAOYSA-N 0.000 description 2
- 208000030090 Acute Disease Diseases 0.000 description 2
- BSYNRYMUTXBXSQ-UHFFFAOYSA-N Aspirin Chemical compound CC(=O)OC1=CC=CC=C1C(O)=O BSYNRYMUTXBXSQ-UHFFFAOYSA-N 0.000 description 2
- 208000014644 Brain disease Diseases 0.000 description 2
- 208000017667 Chronic Disease Diseases 0.000 description 2
- WZUVPPKBWHMQCE-UHFFFAOYSA-N Haematoxylin Chemical compound C12=CC(O)=C(O)C=C2CC2(O)C1C1=CC=C(O)C(O)=C1OC2 WZUVPPKBWHMQCE-UHFFFAOYSA-N 0.000 description 2
- 206010020772 Hypertension Diseases 0.000 description 2
- 229930040373 Paraformaldehyde Natural products 0.000 description 2
- 239000006180 TBST buffer Substances 0.000 description 2
- 208000007536 Thrombosis Diseases 0.000 description 2
- 238000002835 absorbance Methods 0.000 description 2
- 229960001138 acetylsalicylic acid Drugs 0.000 description 2
- 239000012190 activator Substances 0.000 description 2
- 230000001154 acute effect Effects 0.000 description 2
- 230000033115 angiogenesis Effects 0.000 description 2
- 230000003110 anti-inflammatory effect Effects 0.000 description 2
- 210000000702 aorta abdominal Anatomy 0.000 description 2
- 210000001367 artery Anatomy 0.000 description 2
- JAONZGLTYYUPCT-UHFFFAOYSA-K bismuth subgallate Chemical compound OC(=O)C1=CC(O)=C2O[Bi](O)OC2=C1 JAONZGLTYYUPCT-UHFFFAOYSA-K 0.000 description 2
- 229960000199 bismuth subgallate Drugs 0.000 description 2
- 230000017531 blood circulation Effects 0.000 description 2
- 230000030833 cell death Effects 0.000 description 2
- 230000004663 cell proliferation Effects 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- RNFNDJAIBTYOQL-UHFFFAOYSA-N chloral hydrate Chemical compound OC(O)C(Cl)(Cl)Cl RNFNDJAIBTYOQL-UHFFFAOYSA-N 0.000 description 2
- 229960002327 chloral hydrate Drugs 0.000 description 2
- 208000029078 coronary artery disease Diseases 0.000 description 2
- DOBMPNYZJYQDGZ-UHFFFAOYSA-N dicoumarol Chemical compound C1=CC=CC2=C1OC(=O)C(CC=1C(OC3=CC=CC=C3C=1O)=O)=C2O DOBMPNYZJYQDGZ-UHFFFAOYSA-N 0.000 description 2
- 238000007710 freezing Methods 0.000 description 2
- 230000036541 health Effects 0.000 description 2
- 208000019622 heart disease Diseases 0.000 description 2
- 238000007490 hematoxylin and eosin (H&E) staining Methods 0.000 description 2
- 238000003125 immunofluorescent labeling Methods 0.000 description 2
- 230000007365 immunoregulation Effects 0.000 description 2
- 239000012535 impurity Substances 0.000 description 2
- 230000002757 inflammatory effect Effects 0.000 description 2
- 239000007928 intraperitoneal injection Substances 0.000 description 2
- 238000002796 luminescence method Methods 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 230000009707 neogenesis Effects 0.000 description 2
- 229920002866 paraformaldehyde Polymers 0.000 description 2
- 230000002265 prevention Effects 0.000 description 2
- 230000000770 proinflammatory effect Effects 0.000 description 2
- 230000001737 promoting effect Effects 0.000 description 2
- 230000009467 reduction Effects 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 238000007789 sealing Methods 0.000 description 2
- 235000020183 skimmed milk Nutrition 0.000 description 2
- 239000001509 sodium citrate Substances 0.000 description 2
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- 238000010186 staining Methods 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 230000002537 thrombolytic effect Effects 0.000 description 2
- 238000002834 transmittance Methods 0.000 description 2
- 230000002792 vascular Effects 0.000 description 2
- 229960005080 warfarin Drugs 0.000 description 2
- PJVWKTKQMONHTI-UHFFFAOYSA-N warfarin Chemical compound OC=1C2=CC=CC=C2OC(=O)C=1C(CC(=O)C)C1=CC=CC=C1 PJVWKTKQMONHTI-UHFFFAOYSA-N 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- FWBHETKCLVMNFS-UHFFFAOYSA-N 4',6-Diamino-2-phenylindol Chemical compound C1=CC(C(=N)N)=CC=C1C1=CC2=CC=C(C(N)=N)C=C2N1 FWBHETKCLVMNFS-UHFFFAOYSA-N 0.000 description 1
- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 description 1
- 239000005995 Aluminium silicate Substances 0.000 description 1
- 241000255783 Bombycidae Species 0.000 description 1
- 201000006474 Brain Ischemia Diseases 0.000 description 1
- 206010008120 Cerebral ischaemia Diseases 0.000 description 1
- 102000004127 Cytokines Human genes 0.000 description 1
- 108090000695 Cytokines Proteins 0.000 description 1
- SNRUBQQJIBEYMU-UHFFFAOYSA-N Dodecane Natural products CCCCCCCCCCCC SNRUBQQJIBEYMU-UHFFFAOYSA-N 0.000 description 1
- 108090000386 Fibroblast Growth Factor 1 Proteins 0.000 description 1
- 102100031706 Fibroblast growth factor 1 Human genes 0.000 description 1
- 101710088172 HTH-type transcriptional regulator RipA Proteins 0.000 description 1
- 241000238631 Hexapoda Species 0.000 description 1
- 101001046686 Homo sapiens Integrin alpha-M Proteins 0.000 description 1
- 206010020751 Hypersensitivity Diseases 0.000 description 1
- 102100022338 Integrin alpha-M Human genes 0.000 description 1
- CTQNGGLPUBDAKN-UHFFFAOYSA-N O-Xylene Chemical compound CC1=CC=CC=C1C CTQNGGLPUBDAKN-UHFFFAOYSA-N 0.000 description 1
- 239000002033 PVDF binder Substances 0.000 description 1
- 206010063837 Reperfusion injury Diseases 0.000 description 1
- 108010087230 Sincalide Proteins 0.000 description 1
- 208000005392 Spasm Diseases 0.000 description 1
- 108010023197 Streptokinase Proteins 0.000 description 1
- 108090000435 Urokinase-type plasminogen activator Proteins 0.000 description 1
- 102000003990 Urokinase-type plasminogen activator Human genes 0.000 description 1
- 210000000683 abdominal cavity Anatomy 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 230000001464 adherent effect Effects 0.000 description 1
- 239000000556 agonist Substances 0.000 description 1
- 208000026935 allergic disease Diseases 0.000 description 1
- 230000007815 allergy Effects 0.000 description 1
- 235000012211 aluminium silicate Nutrition 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 238000001949 anaesthesia Methods 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 230000002429 anti-coagulating effect Effects 0.000 description 1
- 238000003287 bathing Methods 0.000 description 1
- 230000005540 biological transmission Effects 0.000 description 1
- 230000001914 calming effect Effects 0.000 description 1
- 230000008822 capillary blood flow Effects 0.000 description 1
- 238000010609 cell counting kit-8 assay Methods 0.000 description 1
- 210000003855 cell nucleus Anatomy 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 235000013339 cereals Nutrition 0.000 description 1
- 230000009510 cerebral cortex damage Effects 0.000 description 1
- 206010008118 cerebral infarction Diseases 0.000 description 1
- 238000003759 clinical diagnosis Methods 0.000 description 1
- 208000035850 clinical syndrome Diseases 0.000 description 1
- 230000015271 coagulation Effects 0.000 description 1
- 238000005345 coagulation Methods 0.000 description 1
- 238000007596 consolidation process Methods 0.000 description 1
- 230000008602 contraction Effects 0.000 description 1
- 210000000805 cytoplasm Anatomy 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 230000018044 dehydration Effects 0.000 description 1
- 238000006297 dehydration reaction Methods 0.000 description 1
- 230000006866 deterioration Effects 0.000 description 1
- 229960001912 dicoumarol Drugs 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 208000035475 disorder Diseases 0.000 description 1
- 239000002552 dosage form Substances 0.000 description 1
- 231100000673 dose–response relationship Toxicity 0.000 description 1
- 230000000857 drug effect Effects 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 210000002889 endothelial cell Anatomy 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- YQGOJNYOYNNSMM-UHFFFAOYSA-N eosin Chemical compound [Na+].OC(=O)C1=CC=CC=C1C1=C2C=C(Br)C(=O)C(Br)=C2OC2=C(Br)C(O)=C(Br)C=C21 YQGOJNYOYNNSMM-UHFFFAOYSA-N 0.000 description 1
- DZGCGKFAPXFTNM-UHFFFAOYSA-N ethanol;hydron;chloride Chemical compound Cl.CCO DZGCGKFAPXFTNM-UHFFFAOYSA-N 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 230000003203 everyday effect Effects 0.000 description 1
- 238000000799 fluorescence microscopy Methods 0.000 description 1
- 238000000227 grinding Methods 0.000 description 1
- 230000035931 haemagglutination Effects 0.000 description 1
- 230000003832 immune regulation Effects 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 230000000302 ischemic effect Effects 0.000 description 1
- NLYAJNPCOHFWQQ-UHFFFAOYSA-N kaolin Chemical compound O.O.O=[Al]O[Si](=O)O[Si](=O)O[Al]=O NLYAJNPCOHFWQQ-UHFFFAOYSA-N 0.000 description 1
- 230000002045 lasting effect Effects 0.000 description 1
- 239000012160 loading buffer Substances 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 230000004089 microcirculation Effects 0.000 description 1
- 210000000274 microglia Anatomy 0.000 description 1
- 230000002025 microglial effect Effects 0.000 description 1
- 210000004088 microvessel Anatomy 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004660 morphological change Effects 0.000 description 1
- 210000005036 nerve Anatomy 0.000 description 1
- 210000004940 nucleus Anatomy 0.000 description 1
- 230000002018 overexpression Effects 0.000 description 1
- 230000036407 pain Effects 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 231100000915 pathological change Toxicity 0.000 description 1
- 230000036285 pathological change Effects 0.000 description 1
- 229960001412 pentobarbital Drugs 0.000 description 1
- WEXRUCMBJFQVBZ-UHFFFAOYSA-N pentobarbital Chemical compound CCCC(C)C1(CC)C(=O)NC(=O)NC1=O WEXRUCMBJFQVBZ-UHFFFAOYSA-N 0.000 description 1
- 230000010412 perfusion Effects 0.000 description 1
- 239000006187 pill Substances 0.000 description 1
- 239000013612 plasmid Substances 0.000 description 1
- 230000010287 polarization Effects 0.000 description 1
- 229920002401 polyacrylamide Polymers 0.000 description 1
- 229920002981 polyvinylidene fluoride Polymers 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 230000001376 precipitating effect Effects 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
- 229940043274 prophylactic drug Drugs 0.000 description 1
- 230000000069 prophylactic effect Effects 0.000 description 1
- 238000001742 protein purification Methods 0.000 description 1
- 238000003908 quality control method Methods 0.000 description 1
- 238000010814 radioimmunoprecipitation assay Methods 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- IZTQOLKUZKXIRV-YRVFCXMDSA-N sincalide Chemical compound C([C@@H](C(=O)N[C@@H](CCSC)C(=O)NCC(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(N)=O)NC(=O)[C@@H](N)CC(O)=O)C1=CC=C(OS(O)(=O)=O)C=C1 IZTQOLKUZKXIRV-YRVFCXMDSA-N 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 229960005202 streptokinase Drugs 0.000 description 1
- 238000001356 surgical procedure Methods 0.000 description 1
- 230000009747 swallowing Effects 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
- 229940126585 therapeutic drug Drugs 0.000 description 1
- 235000013619 trace mineral Nutrition 0.000 description 1
- 239000011573 trace mineral Substances 0.000 description 1
- 238000001890 transfection Methods 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- GPRLSGONYQIRFK-MNYXATJNSA-N triton Chemical compound [3H+] GPRLSGONYQIRFK-MNYXATJNSA-N 0.000 description 1
- 229960005356 urokinase Drugs 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
- 239000008096 xylene Substances 0.000 description 1
Images
Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
The invention relates to the technical field of stiff silkworm extracts, and particularly discloses application of a stiff silkworm extract in preparation of a medicine for improving and/or preventing and treating a small cerebrovascular disease and a small cerebrovascular disease-related disease. The stiff silkworm extract is prepared by the steps of extraction treatment, alcohol precipitation treatment and dialysis treatment, wherein the extraction treatment comprises the steps of soaking stiff silkworm in water at 2-5 ℃, heating and extracting to obtain an extracting solution after soaking, the alcohol precipitation treatment comprises the steps of adding absolute ethyl alcohol into the extracting solution to enable the concentration of the ethyl alcohol in the extracting solution to be 65-75%, carrying out alcohol precipitation for 8-14 hours at 2-5 ℃, and heating and evaporating to remove the ethyl alcohol after the alcohol precipitation is finished; the dialysis treatment comprises placing the extractive solution without ethanol into dialysis bag, dialyzing, concentrating, and sterilizing. The invention can prevent and treat CSVD and relative diseases, and the test proves that the effect is better than that of heparin. The preparation method of the stiff silkworm extract is suitable for batch production and has high product yield.
Description
Technical Field
The invention relates to the technical field of stiff silkworm extracts, in particular to a preparation method and application of a stiff silkworm extracting solution.
Background
Cerebrovascular disease (CSVD) IS a clinical syndrome in which microcirculation in the brain IS pathologically caused by various pathogenic factors, and can progress to Ischemic Stroke (IS). Its onset is hidden, and the clinical diagnosis mainly depends on the imaging examination. Aiming at the treatment of the cerebrovascular and small vessel diseases, the principle of improving the blood circulation of cerebral ischemic areas as early as possible and promoting the recovery of nerve functions IS adopted, and thrombolysis or anticoagulation intervention IS usually performed when patients progress to IS, wherein streptokinase and urokinase are mainly adopted for thrombolysis, heparin or dicoumarin and the like are mainly adopted for anticoagulation so as to prevent thrombus extension and new thrombus generation. These drugs are not specific drugs and all have side effects to some extent, so it IS of great significance to explore more effective CSVD prophylactic and therapeutic drugs to achieve intervention before IS onset.
The bombyx batryticatus is a dry body which is killed by infecting larva of 4-5-year-old bombyx mori, an insect in the family Bombycidae, or artificially inoculating white bombyx batryticatus, modern researches show that the bombyx batryticatus mainly contains fat, protein, amino acid, trace elements and the like, the common use method is a bombyx batryticatus decoction piece which plays the roles of calming wind, relieving spasm, dispelling wind, relieving pain, removing blood stasis, dissipating stagnation and the like by decoction or grinding for swallowing, or by oral administration such as pills, powder and the like, however, the bombyx batryticatus is not recorded in CSVD prevention and treatment at present.
Disclosure of Invention
One of the technical problems to be solved by the invention is to provide the application of the stiff silkworm extract in preparing the medicines for improving and/or preventing and treating the small cerebrovascular disease and the diseases related to the small cerebrovascular disease.
The second technical problem to be solved by the invention is to provide the preparation method of the stiff silkworm extract, so that the product yield is high, and the stiff silkworm extract is suitable for preparing medicines for preventing and treating the cerebral small vascular disease and the diseases related to the cerebral small vascular disease.
The technical problem solved by the invention is realized by adopting the following technical scheme:
the application of the stiff silkworm extract and the application of the stiff silkworm extract in preparing the medicines for improving and/or preventing and treating the cerebral small vessel disease and the diseases related to the cerebral small vessel disease.
Further, the stiff silkworm extract is used as a drug effect component and is singly used or combined with a pharmaceutically acceptable excipient to prepare a pharmaceutical preparation in an intramuscular injection administration or intravenous administration dosage form.
Further, the diseases related to the cerebral small vessel diseases comprise ischemic stroke.
The preparation method of the stiff silkworm extract comprises the following steps:
extraction treatment: the white muscardine silkworm is added with water and soaked under the condition of 2-5 ℃, and after soaking, heating extraction is carried out to obtain an extracting solution;
alcohol precipitation treatment: adding absolute ethyl alcohol into the extracting solution to ensure that the concentration of the ethyl alcohol in the extracting solution is 65-75%, carrying out alcohol precipitation for 8-14 h at the temperature of 2-5 ℃, and heating and evaporating to remove the ethyl alcohol after the alcohol precipitation is finished; the control of the alcohol precipitation treatment condition is critical, and improper treatment easily causes excessive impurity protein and has allergy risk.
And (3) dialysis treatment: dialyzing the extractive solution, concentrating the filtrate, and sterilizing.
Further, in the extraction treatment step, the stiff silkworms are taken out before heating and extraction, the soak solution is divided into 2-4 parts, one part of the soak solution is mixed with the stiff silkworms before each heating and extraction, the mixture is boiled and then is decocted by small fire to obtain an extracting solution, and the extracting solutions obtained after multiple heating and extraction are combined.
Further, before the alcohol precipitation treatment, the extracting solution is concentrated, and the mass ratio of the concentrated extracting solution to the stiff silkworm is 2-4:1.
Further, in the alcohol precipitation treatment step, water bath evaporation is carried out at the temperature of 55-65 ℃. The damage and deterioration of the effective components can be caused by overhigh temperature, the efficacy of the product is directly influenced, and the medicine is burnt and deteriorated due to the fact that alcohol is ignited by overhigh temperature.
Further, the dialysis treatment is carried out in a dialysis bag, and the dialysis comprises three times of dialysis, wherein the time of the first dialysis is 1-3 hours, the time of the second dialysis is 5-7 hours, and the time of the third dialysis is 10-14 hours.
Further, the cut-off of the dialysis bag was 5000Da. The product obtained by the interception amount can reserve functional components to the maximum extent, has low impurities, can meet the requirement of preparing injection and can fully exert the effect.
Further, the extract yield after extraction treatment is higher than 44%, the extract yield after alcohol precipitation treatment is higher than 20%, and the extract yield after dialysis treatment is higher than 15%.
Has the advantages that: the medicine prepared from the stiff silkworm extracting solution can prevent and treat CSVD by combining anticoagulation and immunoregulation. Can effectively improve the intracerebral inflammation of a rat of a cerebral small vessel ischemia reperfusion model so as to play a role in neuroprotection and activate the cerebral vascular neogenesis of the rat of the cerebral small vessel ischemia reperfusion model mediated by coagulation Factor XII (FXII).
The stiff silkworm extract can be applied to preparation of IS prevention and treatment medicines and has better effect than heparin.
The preparation method of the stiff silkworm extract is suitable for batch production, production nodes are easy to control, product quality is stable, product yield is high, and the prepared stiff silkworm extract has a good effect of improving and/or preventing and treating the small cerebrovascular disease and the diseases related to the small cerebrovascular disease.
Drawings
FIG. 1 shows the effect of Bombyx Batryticatus extract on the morphology of the cerebral cortex of I/R rats (HE staining, X100). ( control: blank group; model control is a Model group; positive control heparin group; L-DG: low dose group of Bombyx Batryticatus extract; M-DG: preparing a middle-dose group of stiff silkworm extract; H-DG: bombyx Batryticatus extractive solution high dose group. )
FIG. 2 shows the results of experiments on the effect of Bombyx Batryticatus extract on the brain kallikrein release system and angiogenesis in ischemia-reperfusion rat brain tissue (
n = 3). (wherein part A indicates the overall result, FXII and KK belong to the kallikrein system, VEGF is an endothelial cell growth factor promoting angiogenesis, CD31+ and Brdu +/vWF + belong to the neovascular surface marker, part B indicates the result of immunofluorescence assay of KK, part C indicates the result of immunofluorescence assay of CD31, and panel D indicates the result of immunofluorescence assay of Brdu +/vWF +. The P < 0.05% in comparison to the blank group(ii) a * Denotes P < 0.01 compared to the blank group; Δ represents P < 0.05 compared to model group). ( control: blank group; model control is a Model group; positive control heparin group; L-DG: low dose group of Bombyx Batryticatus extract; M-DG: preparing a middle-dose group of stiff silkworm extract; H-DG: bombyx Batryticatus extractive solution high dose group. )
Fig. 3 shows the morphology of brain microvascular endothelial cells in rats in the oxygen deprivation model (n = 3).
FIG. 4 shows the proliferation of brain microvascular endothelial cells in rats in an oxygen-deprivation model (
n = 3) (a) proliferation of microvascular endothelial cells. And (B) VEGF expression of microvascular endothelial cells. control: blank group; plasma: a blank plasma group; BB plasma: bombyx Batryticatus extractive solution and blood plasma group; BB-mediated plasma: bombyx Batryticatus extractive solution containing medicinal plasma group; bs: a Bismuth subgallate positive drug group; positive: a heparin group; and (3) a Serum: a serogroup; BB: bombyx Batryticatus extractive solution group; IG: BKR2 inhibitor group. * Represents P < 0.05 compared to the blank group; * Denotes P < 0.01 compared to the blank group; Δ represents P < 0.05 compared to the model group.
Detailed Description
In order to make the technical means, the creation features, the achievement purposes and the effects of the invention easy to understand, the invention is further described with the specific embodiments.
Example 1
The preparation method of the stiff silkworm liquid comprises the following steps:
extraction treatment: weighing Bombyx Batryticatus 100.0g, adding 1L water, and soaking at 4 deg.C for 10 hr; the stiff silkworm is taken out before heating, the soak solution is divided into 3 parts, one part of soak solution is mixed with the stiff silkworm before each heating extraction, the mixture is boiled and then is decocted by small fire to obtain an extracting solution, and the extracting solution is combined after the three times of decoction extraction.
Alcohol precipitation treatment: concentrating the mixed extractive solution to 300ml, adding anhydrous ethanol into the concentrated extractive solution to make ethanol concentration in the extractive solution 70%, precipitating with ethanol at 4 deg.C for 10 hr, and evaporating in water bath at 60 deg.C to remove ethanol;
and (3) dialysis treatment: putting the extract without ethanol into a dialysis bag with a cut-off of 5000Da for dialysis treatment, wherein the dialysis treatment comprises three times of dialysis in sequence, the three times of dialysis are 2h, 6h and 12h in sequence, after the dialysis is finished, the extract is concentrated to 100ml, and after autoclaving treatment, the extract is prepared into a product with the concentration of 1g/ml, and the product is sealed and packaged and then stored at 4 ℃ for later use.
In this example, the extract yield after the extraction treatment was 44.2%, the extract yield after the alcohol precipitation treatment was 20.9%, and the extract yield after the dialysis treatment was 15.1%.
Example 2
The application of the stiff silkworm extract obtained in the embodiment 1 in preparing medicines for preventing and treating cerebral small vessel diseases.
Rat administration dose = human administration dose × conversion factor =10g · 70kg in terms of human dose and mouse conversion -1 ·d -1 ×6.3≈0.9mg/g -1 ·d -1 To obtain the high dose, the medium dose and the low dose of the muscardine silkworm extract which are respectively 1.8mg/g -1 ·d -1 、0.9mg/g -1 ·d -1 、0.45mg/g -1 ·d -1 。
1. Materials and methods
1.1 study subjects: 60 male SPF-level SD rats of 200-220 g were purchased from Schlekschada, inc. in Hunan, and after 24 hours of adaptive feeding, were randomly grouped as: sham operation group (0.9% NaCl), model group (0.9% NaCl), positive drug (heparin) group (150 IU), and Bombyx Batryticatus extract low dose group (0.45 mg/g) -1 ·d -1 ) And the dosage group in the stiff silkworm extract (0.9 mg/g) -1 ·d -1 ) Bombyx Batryticatus extractive solution high dose group (1.8 mg/g) -1 ·d -1 ) Before 4 days and after 3 days, the corresponding medicine is continuously injected into the abdominal cavity for 1 time every day, and the total time is 7 days.
A model for treating small cerebrovascular diseases, namely a rat I/R model for repeatedly clamping and recanalizing bilateral common carotid arteries. 25 percent of urethane and 10 percent of chloral hydrate 1:1 are compounded for intraperitoneal injection anesthesia, and bilateral common carotid arteries are separated. Under a small animal speckle imager, the small animal speckle imager is clamped by an artery clamp for 1.5min and then is released for 1min, after 5 times of continuous operation, the blood flow of visible micro-vessels in the imager is reduced by 30%, and the skin is sutured. The sham operation group only sews the skin after separating the common carotid arteries on both sides, and does not perform the clipping and recanalization operation.
1.2 rat behavioural assays
BBT is the classic behavioural model of animal motor ability reduction or cerebral cortex damage research, and during the test, put the rat on 1.5cm wide stuff, one end is unsettled, and one end is fixed in 40cm x 40cm flat center, records rat 2min test time internal balance ability, and the standard of grading: standing stably on the batten without shaking, and recording for 1min after 2 min; standing stably on the batten, shaking left and right without sliding down, and recording for 2 minutes after lasting for 2 min; standing on the batten, sliding to one side, not dropping, and recording for 3 minutes after 2 min; standing on the batten for less than 2min, and taking 4 minutes from the batten; trying to stand stably on the batten, but taking 5 minutes after falling for several seconds; no standing ability was scored 6 points.
1.3 animal treatment and materials selection
After the evaluation of rat BBT, 4 animals per group were randomly selected, 25% of urethane and 10% of chloral hydrate 1:1 were mixed for intraperitoneal injection and anaesthesia, the brain was taken after 4% of paraformaldehyde heart perfusion, and paraffin sections were taken for HE staining and immunofluorescence staining. The remaining rats in each group were harvested for fresh brain tissue, isolated in Cerebral Cortical (CC) regions, placed in different EP tubes and stored at-80 ℃ for ELISA and non-target metabonomics detection.
1.4HE staining
The paraffin blocks are taken, brain slices are cut, the paraffin blocks are respectively dyed by 1% eosin and 0.2% hematoxylin dye, and after color separation by 1% hydrochloric acid ethanol, images are collected by a common microscope.
1.5 immunofluorescence staining
Paraffin blocks were taken, brain sections were dissected, 0.5% Triton membrane rupture, 5% BCA block, primary anti-CD 45+, CD11b +, arg-1+ (1, 500 dilution) overnight at 4 ℃, primary antibody was discarded, PBS rinsed 3 times, secondary antibody (1, 1 000) was incubated at 37 ℃ for 1h, dapi coverslips, and images were collected by fluorescence microscopy.
1.6 detection of the expression of the brain cortex cytokines by ELISA
Adding RIPA into rat cerebral cortex, homogenizing on ice, carrying out ultrasonic disruption, extracting total protein, detecting absorbances of inflammatory factors IL-1 beta, IL-6, IL-4 and IL-10 of brain tissues under the wavelength of 450nm according to the steps of an ELISA specification, and calculating the content of each inflammatory factor in a sample according to a standard curve.
1.7 four tests of in vitro intervention of Bombyx Batryticatus extract on human plasma coagulation
Whole blood (standard of inclusion: 20-65 years old, no acute and chronic diseases such as cold, hypertension, coronary heart disease and the like, and no administration history of anti-freezing drugs such as aspirin, warfarin and the like) of healthy people comes from the health management department of the first subsidiary hospital of Hunan Chinese medicine university. The plasma is prepared by centrifugation in a laboratory for preventing and treating the heart and brain diseases by combining Chinese traditional medicine and western medicine of the university of traditional Chinese medicine in Hunan, and is divided into a blank group (0.9 percent NaCl), a low-dose group (3 mg. ML-1) of the stiff silkworm extract, a medium-dose group (6 mg. ML-1) of the stiff silkworm extract and a high-dose group (9 mg. ML-1) of the stiff silkworm extract, and the plasma and the intervention components 1:1 of each group are mixed and then are processed by a full-automatic hemagglutination instrument to detect PT, APTT, TT and FIB.
1.8 non-target metabonomics detection metabolite change test of Bombyx Batryticatus extract
The batryticated silkworm extract, the false operation group, the model group and the dosage group rat plasma and cerebral cortex tissue in the batryticated silkworm extract are sent to Beijing Nuo cereal induced science and technology company, and non-target metabonomics detection is carried out.
Comparing the medicated blood plasma of Bombyx Batryticatus with the blood plasma of model group to find new metabolites with remarkably increased content; comparing the brain cortex of the rat with the brain cortex of the model group by the intervention of the bombyx batryticatus, and finding new addition and content remarkably increased metabolism; comparing newly increased and obviously increased metabolites of the plasma and the cerebral cortex of rats in the batryticated silkworm intervention group, and finding the newly increased and obviously increased metabolites of the content; compared with newly added rats and cerebral cortex in the stiff silkworm extract and stiff silkworm dry-treatment group, the metabolite content is obviously increased.
2. Test results
2.1 Effect of Bombyx Batryticatus extract on mouse behaviourology
The result shows that the stiff silkworm extract can obviously reduce the behavioral score of the I/R model rat with the cerebrovascular disease. As shown in table 2, the BBT score was increased in model group rats on days 1 and 3, compared to the sham-operated group; compared with the model group, the dry prognosis of the batryticated silkworm extract solution with low, medium and high dose is obviously reduced by the BBT score of the I/R rat on the 1 st and 3 rd days, and the difference of the positive medicament (heparin) group has no statistical significance.
TABLE 1 influence of Bombyx Batryticatus extract on I/R rat behaviourology
n＝4)
Note: comparison with sham group 1) P＜0.05， 2) P is less than 0.01; comparison with model group 3) P＜0.05， 4) P＜0.01。
2.2 Effect of Bombyx Batryticatus extract on morphology of cerebral cortex of I/R rat
The results are shown in FIG. 1, in the sham-operated group, the cerebral cortex cells were well-arranged, the nuclei were rounded, and the cytoplasm was full; the model group can see a large number of abnormal cells, the arrangement is loose and disordered, part of cell nuclei are solidified and shrunk, the cell gaps are widened, and vacuole-like changes occur; the positive drug (heparin) group did not improve significantly; after the medicine dry prognosis is carried out on the batryticated silkworm extract, the degree of cell transportation disorder is obviously reduced, the nuclear consolidation and vacuole-like cell quantity is obviously reduced, and the batryticated silkworm extract can obviously improve the cerebral cortex morphology of rats of I/R model of the cerebral small vessel disease.
2.3 Effect of Bombyx Batryticatus extract on I/R rat brain tissue blood-borne immune cell infiltration
The results are shown in table 2, and compared with the sham operation group, the model group CC brain area CD45 fluorescence intensity is obviously increased; compared with the model group, after the dry drug prognosis is carried out on the batryticated silkworm extract, the CD45 fluorescence intensity of the batryticated silkworm extract in the high dose group is reduced, although the CD45 fluorescence intensity of the batched silkworm extract in the low dose group is slightly reduced, the difference has no statistical significance, and the batched silkworm extract in the middle dose and the high dose can obviously reduce the brain tissue blood-borne immune cell infiltration of I/R model rats with the cerebral small vessel diseases and promote the microglial cells to be polarized to M2 type, thereby having obvious positive influence on the immune regulation of organisms.
TABLE 2 Bombyx Batryticatus extract on average against CD45 in CC brain region of I/R ratInfluence of fluorescence intensity: (
n＝6)
2.4 Effect of Bombyx Batryticatus extract on I/R rat CC brain region inflammation
The results are shown in Table 3, compared with the sham operation group, the model group rat CC brain area proinflammatory factor IL-1 beta and IL-6 protein expression level is increased, and the anti-inflammatory factor IL-4 and IL-10 protein expression level is reduced; compared with the model group, the low, medium and high dose groups of the batryticated silkworm extract liquid CC brain area proinflammatory factors IL-1 beta and IL-6 are averagely reduced, the anti-inflammatory factors IL-4 and IL-10 are averagely increased, the difference of the positive medicament (heparin) group has no statistical significance, which shows that the bated silkworm extract liquid obviously relieves the inflammatory reaction in the brain tissue of the I/R model rat with the cerebrovascular disease and plays a role in neuroprotection.
TABLE 3 influence of Bombyx Batryticatus extract on the expression of IL-1 beta, IL-6, IL-4, IL-10 proteins in CC brain region of I/R rat: (
n＝9)(pg·mL-1)
2.5 Effect of Bombyx Batryticatus extract on anticoagulation
The results are shown in Table 4. Compared with the blank group, the human plasma APTT, PT and TT of the batryticated silkworm extract intervention group is obviously prolonged and is dose-dependent, and the difference has statistical significance, but has no influence on FIB and has no statistical significance.
TABLE 4 anticoagulant effect quality control of Bombyx Batryticatus extractive solution: (
n＝4)
Note: comparison with sham surgery group 1) P＜0.05， 2) P＜0.01。
2.6 non-target metabonomics detection results of Bombyx Batryticatus extract
The detection shows that 809 metabolites are identified in the stiff silkworm extract, 137 metabolites are identified in the stiff silkworm extract after the stiff silkworm extract intervenes in the plasma of the I/R rat, 172 metabolites are identified in the brain tissue of the I/R rat, and compared with the plasma and the brain tissue of the I/R rat, 57 metabolites enter the blood and 45 metabolites enter the brain in the stiff silkworm extract. After the effect of the stiff silkworm extract, the metabolites entering blood and brain are compared, and the total number of the 15 common metabolites is 15, and the 15 common metabolites are shown in a table 5.
TABLE 5 common metabolites of Bombyx Batryticatus extract entering brain and blood: (
n＝3)
In conclusion, the stiff silkworm extract has a remarkable anticoagulation effect, can reduce the behavioral scores of rats with I/R models of the cerebral small vascular diseases, improve the morphology of cerebral cortex of rats with I/R models of the cerebral small vascular diseases, reduce the blood-borne immune cell infiltration of the cerebral tissues of rats with I/R models of the cerebral small vascular diseases, promote the polarization of microglia to M2 type, remarkably relieve the inflammatory reaction in the cerebral tissues of rats with I/R models of the cerebral small vascular diseases to play a role in neuroprotection, prevent and improve CSVD by combining anticoagulation and immunoregulation, can be expected to effectively improve the clinical symptoms of IS patients, and has a better effect than heparin.
Example 3
The application of the stiff silkworm extract obtained in the embodiment 1 in preparing a medicament for preventing and treating small cerebral vascular diseases in improving cerebral ischemia-reperfusion injury.
Through the conversion of the human medicine dose and the mouse, the rat administration dose = the human administration dose multiplied by the conversion coefficient =10g 70kg-1 d-1 x 6.3 ≈ 0.9mg/g-1 d-1, and the high dose, the medium dose and the low dose of the muscardine silkworm extract are respectively 1.8mg/g -1 ·d -1 、0.9mg/g -1 ·d -1 、0.45mg/g -1 ·d -1 。
1. Test method
1.1 plasma preparation
1.1.1 preparation of human plasma
The whole blood of healthy people (brought into the standard of 20-65 years old, without acute and chronic diseases such as cold, hypertension, coronary heart disease and the like, and without the taking history of anti-freezing drugs such as aspirin, warfarin and the like) comes from the health management department of the first subsidiary hospital of the Hunan Chinese medicinal university. Centrifugating in a laboratory for preventing and treating heart and brain diseases in combination of Chinese and western medicine of Hunan university of traditional Chinese medicine to obtain blood plasma, subpackaging, and storing at-80 deg.C (within 6 months).
1.1.2 preparation of animal serum and plasma blanks
After 12h of adaptive SD feeding of 4-week-old male, ip 1mL of normal saline, 1 time/d, continuous 7d, 6h after the last treatment, ip 3% sodium pentobarbital (1 mL/kg) for anesthesia, abdominal aorta blood sampling, partial whole blood filling into a 5mL blank tube, partial whole blood filling into a 2mL sodium citrate blood sampling tube, numbering, 3500r/min (serum standing overnight at 4 ℃) for 10min to obtain rat blank serum and plasma, subpackaging and storing at-80 ℃.
1.1.3 preparation of plasma containing drugs in animals
After 12h of adaptive feeding of 4-week-old male SD rats, ip 1mL of stiff silkworm extract (105 mg/L), 1 time/d, and 7d continuously, 6h after last administration, ip 25% urethane (4 mL/kg) anesthesia, abdominal aorta blood sampling, whole blood filling into a 2mL sodium citrate blood sampling tube, numbering, 3500r/min centrifugation for 10min to obtain rat drug-containing plasma, and subpackaging and storing at-80 ℃.
1.2 HEK-293 cell preparation of overexpression of FXII protein
The HEK-293 cell is transferred into FXII plasmid according to the operation of the nuclear transfer kit instruction, cultured for 24h under the conditions of 37 ℃ and 5% CO2, the transfection rate can reach 95%, and the HEK-293 cell for stably expressing FXII protein is obtained by screening with G418. Collecting cell supernatant, loading the cell supernatant into a protein purification column at 10 mu L/s, and eluting with 0.25nmol/L imidazole to obtain FXII purified protein.
1.3FXII activation assay
Adding 40 μ L of 1 μ g/. Mu.L Bombyx Batryticatus extractive solution into FXII protein-containing cell suspension (medical physiological saline for blank group, and agonist: 3.94 × 10) -6 Mu.g/. Mu.L Bismuth subgallate), incubating for 5min at 37 ℃, adding 2 xSDS loading buffer and 2.5% beta-ME, after bathing for 5min in hot water, loading 10% acrylamide gel, 12. Mu.L/well, 100V running gel, after wet-rotating for 2h at 120V, sealing for 1h in skimmed milk, adding FXII antibody, incubating overnight at 4 ℃, washing membranes by TBST, adding secondary antibody, incubating for 1h at 37 ℃, and developing on a Bio-Rad ChemiDocTM imaging system by an ECL luminescence method.
1.4KK Generation experiment
Taking 20 mu L of human plasma, treating a sample according to an FXII activation experiment method, loading the sample on 8% acrylamide gel, 30 mu L/hole, 100V running gel, sealing in skimmed milk for 1h after 120V wet-conversion for 2h, adding a KK antibody, incubating overnight at 4 ℃, washing a membrane with TBST, adding a second antibody, incubating for 1h at 37 ℃, and developing by an ECL luminescence method in a Bio-Rad ChemiDocTM imaging system.
Adding 20 μ L of human whole plasma or plasma lacking FXII into each well of 96-well plate, respectively adding 40 μ L of 1 μ g/μ L of Bombyx Batryticatus extractive solution, using 40 μ L of physiological saline as blank control group, and 40 μ L of kaolin solution as positive control group. After incubation at 37 ℃ for 5min, 20. Mu.L of 5mg/L S-2302 was added and incubated at 37 ℃ for 30min on a microplate reader, and the absorbance (A) at 405nm was measured every 5 minutes.
1.5 preparation of animal models
After adaptive feeding of 30 SD rats for 12h, the SD rats are randomly divided into a blank group (normal saline), a model group (normal saline), a heparin group (150 IU) and a stiff silkworm low, medium and high dose group, wherein the weight ratio of the SD rats is calculated according to the dosage of human bodies: the dosage of the low, medium and high dose groups is 0.45mg/g/d, 0.9mg/g/d and 1.8mg/g/d respectively. Except for the blank group, the bilateral common carotid arteries of the other rats were isolated, and under a small animal speckle imager, the rats were clamped by an artery clamp for 1.5min and then released for 1min, and after 5 times of continuous operation, the obvious capillary blood flow reduction (30 +/-5)%, which was observed in the imager, was sutured to the skin.
1.6 rat brain tissue index detection
Fixing each group of rat brain tissues by 4% paraformaldehyde, embedding by steps of ethanol dehydration, xylene transparency and the like to prepare paraffin blocks, slicing, and staining by hematoxylin-eosin (HE) to observe pathological changes of the brain tissues; KK, CD31, brdu/vWF antibodies were incubated, observed under a microscope and photographed.
Taking rat brain tissue, homogenizing on ice, extracting protein, measuring the protein concentration by using a BCA method, carrying out electrophoresis on a protein sample by using 10% sodium dodecyl sulfate-polyacrylamide gel, transferring to a PVDF membrane, incubating FXII, KK and VEGF antibodies and a second antibody, and developing.
1.7 preparation of an oxygen sugar deprivation model
Rat brain microvascular endothelial cells were divided into a blank group (10 μ L physiological saline), a blank plasma group (10% blank plasma), a Bombyx Batryticatus plasma group (10% 26.25mg/L Bombyx Batryticatus extract +10% blank plasma), a Bombyx Batryticatus drug-containing plasma group (10% Bombyx Batryticatus extract-containing plasma), a heparin group (10 IU heparin +10% blank plasma), an activator group (10 nmol/L Bismuth sublallate +10% blank plasma), a blank serum group (10% blank serum), a BKR2 inhibitor group (15 nmol/L Ibatiabant Acetate +10% Bombyx Batryticatus extract-containing plasma), cultured for 12 hours under the conditions of 5% CO2 and 1% O2 using a sugar-free medium, and after 30min of reoxygenation, observed and Western photographed under a microscope, cell proliferation was detected by the CCK-8 method, and expression of VEGF protein in the cells was detected by the blotting method.
2. Analysis of results
2.1 Effect of Bombyx Batryticatus extract on brain kallikrein release system of ischemia reperfusion rat brain tissue
As shown in part A of FIG. 2, FXII protein activation was not evident in the model group and heparin group, and KK and VEGF protein expression was significantly increased (P < 0.05) compared to the blank group. Compared with the model group, the FXII prototype protein expression of the stiff silkworm extract group is obviously reduced (P is less than 0.05), and dose correlation is realized, so that a large amount of protein is prompted to be activated; KK and VEGF protein expression were further elevated (P < 0.05), dose-related.
KK expression was increased in each group (P < 0.05) compared to the blank group, but Bombyx Batryticatus extract group was better and dose-related as shown in section B, C, D of FIG. 2. In immunofluorescence assay, model group, heparin group CD31, compared to blank group + And Brdu + /vWF + No obvious increase is seen; bombyx Batryticatus extractive solution group CD31 and Brdu compared with model group + /vWF + Has obvious expression (P < 0.05, 0.01) and dose correlation.
2.2 Effect of Bombyx Batryticatus extract on morphological changes of brain microvascular endothelial cells of rats in model of oxygen deprivation
As shown in figure 3, after the simultaneous modeling, the cells of each group are changed, the cell contraction and the light transmittance are changed, the blank plasma group and the FXII activator group have obvious fibrin generation, and obvious fibrin lumps and fibrin filaments are seen under the microscope. Although no obvious fibrin clot is seen in the blank group, the heparin group, the serum group containing the medicine and the BKR2 inhibitor group, the number of adherent cells is obviously reduced, the solid shrinkage is obvious, the light transmittance is poor, and the cell proliferation is poor. Under the joint intervention of the stiff silkworm extract and the rat plasma, no obvious fibrin lumps are seen, the cell number of the stiff silkworm extract and the rat plasma is also obviously higher than that of the plasma group, but partial cell death and solid shrinkage phenomena can be seen. The cell state of the plasma group containing the batryticated silkworm extract is obviously better than that of other treatment groups, the cell light transmission is strong, and no obvious cell death and form change are seen.
2.3 Effect of Bombyx Batryticatus extract on proliferation of brain microvascular endothelial cells in rats in model of oxygen deprivation
As shown in figure 4, compared with the plasma group, endothelial cells of the heparin group, the stiff silkworm extract and the plasma joint drying group and the stiff silkworm extract-containing plasma group are obviously proliferated, wherein the effect of the stiff silkworm extract-containing plasma group is most obvious, and the difference has statistical significance (P is less than 0.01). However, compared with the blank group and the plasma group, the expression of VEGF protein is obviously increased in the stiff silkworm extract combined plasma group and the stiff silkworm-containing plasma group (P is less than 0.05).
In conclusion, the stiff silkworm extract can effectively improve the intracerebral inflammation of the cerebral small vessel ischemia reperfusion model rat so as to play a role in neuroprotection, and can activate the blood coagulation Factor XII (FXII) mediated cerebral small vessel ischemia reperfusion model rat cerebral vascular neogenesis.
The foregoing shows and describes the general principles, essential features, and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are described in the specification and illustrated only to illustrate the principle of the present invention, but that various changes and modifications may be made therein without departing from the spirit and scope of the present invention, which fall within the scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.
Claims (10)
1. The application of the stiff silkworm extract is characterized in that the stiff silkworm extract is applied to the preparation of medicines for improving and/or preventing and treating the small cerebrovascular disease and the diseases related to the small cerebrovascular disease.
2. The use as claimed in claim 1, wherein the stiff silkworm extract is used as a pharmaceutical effective ingredient alone or in combination with pharmaceutically acceptable excipients to prepare a pharmaceutical preparation in the form of intramuscular injection or intravenous administration.
3. The use of claim 1, wherein the disease associated with cerebrovascular and cerebrovascular disorders comprises ischemic stroke.
4. The method for preparing the stiff silkworm extract solution according to claim 1, wherein the method for preparing the stiff silkworm extract solution comprises the following steps:
extraction treatment: the white muscardine silkworm is soaked in water at the temperature of 2-5 ℃, and after the soaking is finished, the white muscardine silkworm is heated and extracted to obtain an extracting solution;
alcohol precipitation treatment: adding absolute ethyl alcohol into the extracting solution to ensure that the concentration of the ethyl alcohol in the extracting solution is 65-75%, carrying out alcohol precipitation for 8-14 h at the temperature of 2-5 ℃, and heating and evaporating to remove the ethyl alcohol after the alcohol precipitation is finished;
and (3) dialysis treatment: dialyzing the extractive solution, concentrating the filtrate, and sterilizing.
5. The method for preparing Bombyx Batryticatus extract according to claim 4, wherein in the step of extraction treatment, bombyx Batryticatus is taken out before heating and extracting, the soaking solution is divided into 2-4 parts, one part of soaking solution is mixed with Bombyx Batryticatus before each heating and extracting, the mixture is boiled and decocted with small fire to obtain extract, and the extract obtained after multiple heating and extracting is combined.
6. The method for preparing Bombyx Batryticatus extract according to claim 4, wherein before the ethanol precipitation treatment, the extract is concentrated, and the mass ratio of the concentrated extract to Bombyx Batryticatus is 2-4:1.
7. The method for preparing Bombyx Batryticatus extract according to claim 4, wherein the ethanol precipitation step is carried out by evaporation in a water bath at 55-65 ℃.
8. The preparation method of Bombyx Batryticatus extract according to claim 4, wherein the dialysis treatment is carried out in a dialysis bag, the dialysis comprises three times of dialysis, the first time of dialysis is 1-3 h, the second time of dialysis is 5-7 h, and the third time of dialysis is 10-14 h.
9. The method of preparing Bombyx Batryticatus extract according to claim 8, wherein the cut-off volume of the dialysis bag is 5000Da.
10. The method of claim 4, wherein the yield of the extract obtained by the extraction is higher than 44%, the yield obtained by the alcohol precipitation is higher than 20%, and the yield obtained by the dialysis is higher than 15%.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211469580.XA CN115778985A (en) | 2022-11-22 | 2022-11-22 | Preparation method and application of stiff silkworm extract |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211469580.XA CN115778985A (en) | 2022-11-22 | 2022-11-22 | Preparation method and application of stiff silkworm extract |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN115778985A true CN115778985A (en) | 2023-03-14 |
Family
ID=85440210
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202211469580.XA Pending CN115778985A (en) | 2022-11-22 | 2022-11-22 | Preparation method and application of stiff silkworm extract |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN115778985A (en) |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105169216A (en) * | 2015-07-20 | 2015-12-23 | 冉宇欣 | Pharmaceutical preparation for treating transient ischemic attack with syndrome of blood stasis in orifices and preparation method of pharmaceutical preparation |
| CN108904617A (en) * | 2018-10-11 | 2018-11-30 | 刘国荣 | The pharmaceutical composition and its preparation method and application for treating cranial vascular disease |
-
2022
- 2022-11-22 CN CN202211469580.XA patent/CN115778985A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105169216A (en) * | 2015-07-20 | 2015-12-23 | 冉宇欣 | Pharmaceutical preparation for treating transient ischemic attack with syndrome of blood stasis in orifices and preparation method of pharmaceutical preparation |
| CN108904617A (en) * | 2018-10-11 | 2018-11-30 | 刘国荣 | The pharmaceutical composition and its preparation method and application for treating cranial vascular disease |
Non-Patent Citations (5)
| Title |
|---|
| "怎样使用僵蚕和佛手?-----中药的使用", 百度, 30 November 2017 (2017-11-30), pages 1 * |
| 彭延古;李露丹;雷田香;付灿云;罗跃;: "僵蚕抗凝成分对血小板聚集的抑制效应", 血栓与止血学, no. 02, 20 April 2007 (2007-04-20) * |
| 雷田香: "僵蚕纯化液抗血栓作用机理的研究", 《中国优秀硕士学位论文全文数据库医药卫生科技辑》, no. 5, 15 May 2010 (2010-05-15), pages 1 * |
| 雷田香等: "僵蚕纯化液抗血栓作用机理的研究", 《中国优秀硕士学位论文全文数据库 医药卫生科技辑》, no. 5, 15 May 2010 (2010-05-15) * |
| 黄晓松;罗银利;于春艳;谭李红;易艳辉;: "僵蚕天南星方对大鼠急性缺血性脑水肿MMP-2表达的影响", 现代中西医结合杂志, no. 16, 1 June 2016 (2016-06-01) * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Li et al. | Caspase-1 inhibition mediates neuroprotection in experimental stroke by polarizing M2 microglia/macrophage and suppressing NF-κB activation | |
| Zekki et al. | The clinical course of experimental autoimmune encephalomyelitis is associated with a profound and sustained transcriptional activation of the genes encoding toll‐like receptor 2 and CD14 in the mouse CNS | |
| US20190269609A1 (en) | A multi-component injection | |
| AU2016202674A1 (en) | Compositions and methods for prevention and treatment of brain diseases and conditions | |
| Liu et al. | Dendrobium alkaloids promote neural function after cerebral ischemia–reperfusion injury through inhibiting pyroptosis induced neuronal death in both in vivo and in vitro models | |
| JP2010504083A5 (en) | ||
| CN113493491B (en) | Polypeptide for preventing or treating ulcerative colitis | |
| JP2607751B2 (en) | Treatment and prevention of autoimmune uveitis | |
| Ma et al. | Mingmu Xiaoyao granules regulate the PI3K/Akt/mTOR signaling pathway to reduce anxiety and depression and reverse retinal abnormalities in rats | |
| JP7002788B2 (en) | Pharmaceutically acceptable salts of polypeptides and their use | |
| KR101667224B1 (en) | Pharmaceutical compositions for combating thrombotic diseases and their preparation and uses | |
| CN108586582B (en) | Anticoagulation polypeptide FX18 and application thereof | |
| CN110339232A (en) | A kind of Chinese medicine composition prevented and/or treat ischemical reperfusion injury | |
| CN106963803B (en) | Application of gynostemma pentaphylla total flavone in preparing medicine for preventing and treating cardiac hypertrophy | |
| JP7073486B2 (en) | Peptide composition for the treatment of damage associated with excitatory neurotoxicity | |
| CN110755450B (en) | Application of extracellular vesicles derived from mesenchymal stem cells in treatment of subarachnoid hemorrhage | |
| CN110025664B (en) | Pharmaceutical composition for preventing and treating cerebral infarction and/or vascular dementia and application thereof | |
| CN116019813B (en) | Use of Vesatolimod in the preparation of a medicament for the prophylaxis and/or treatment of disorders of the central nervous system | |
| JP4463885B2 (en) | Fulminant hepatitis disease treatment | |
| CN115778985A (en) | Preparation method and application of stiff silkworm extract | |
| KR100950164B1 (en) | Healthy functional food and pharmaceutical composition for prevention and treatment of stroke and neurodegenerative diseases including dementia | |
| JPH1036388A (en) | Synthesis inhibitor containing ginsenoside of protein belonging to hsp 27 family | |
| WO2021249402A1 (en) | Effects of cell-free fat liquid extract on macrophage polarization modulation and disease treatment | |
| WO2009135423A1 (en) | Pharmceuttical composition for treating cardio-cerebro vascular diseases and preparative method and kit thereof | |
| US20070160701A1 (en) | Hemostatic mistura of ipomoea balatas leaves, methods of preparation and use thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |