CN1895300A - Kosam extract, its extraction and use - Google Patents
Kosam extract, its extraction and use Download PDFInfo
- Publication number
- CN1895300A CN1895300A CN 200610014349 CN200610014349A CN1895300A CN 1895300 A CN1895300 A CN 1895300A CN 200610014349 CN200610014349 CN 200610014349 CN 200610014349 A CN200610014349 A CN 200610014349A CN 1895300 A CN1895300 A CN 1895300A
- Authority
- CN
- China
- Prior art keywords
- fructus bruceae
- lixiviate
- extract
- virus
- ratio
- 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 87
- 238000000605 extraction Methods 0.000 title description 7
- 241000700605 Viruses Species 0.000 claims abstract description 39
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 20
- 241000712461 unidentified influenza virus Species 0.000 claims abstract description 19
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims abstract description 18
- 239000011347 resin Substances 0.000 claims abstract description 16
- 229920005989 resin Polymers 0.000 claims abstract description 16
- 239000006228 supernatant Substances 0.000 claims abstract description 12
- 238000010438 heat treatment Methods 0.000 claims abstract description 8
- 241000701161 unidentified adenovirus Species 0.000 claims abstract description 8
- 239000009969 fructus bruceae Substances 0.000 claims description 104
- 238000000034 method Methods 0.000 claims description 22
- IDGUHHHQCWSQLU-UHFFFAOYSA-N ethanol;hydrate Chemical compound O.CCO IDGUHHHQCWSQLU-UHFFFAOYSA-N 0.000 claims description 20
- 230000002829 reductive effect Effects 0.000 claims description 20
- 241000709661 Enterovirus Species 0.000 claims description 19
- UGAPHEBNTGUMBB-UHFFFAOYSA-N acetic acid;ethyl acetate Chemical compound CC(O)=O.CCOC(C)=O UGAPHEBNTGUMBB-UHFFFAOYSA-N 0.000 claims description 11
- 238000010828 elution Methods 0.000 claims description 11
- 239000002024 ethyl acetate extract Substances 0.000 claims description 11
- 241000238370 Sepia Species 0.000 claims description 10
- 239000003480 eluent Substances 0.000 claims description 10
- 238000002360 preparation method Methods 0.000 claims description 7
- 238000010298 pulverizing process Methods 0.000 claims description 5
- 239000003443 antiviral agent Substances 0.000 claims description 3
- 239000007788 liquid Substances 0.000 abstract description 23
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 abstract description 4
- 235000013399 edible fruits Nutrition 0.000 abstract description 3
- 238000001914 filtration Methods 0.000 abstract description 2
- 241000158621 Brucea Species 0.000 abstract 2
- 239000007864 aqueous solution Substances 0.000 abstract 1
- 125000004122 cyclic group Chemical group 0.000 abstract 1
- 230000000968 intestinal effect Effects 0.000 abstract 1
- 210000004027 cell Anatomy 0.000 description 27
- 239000003814 drug Substances 0.000 description 17
- 230000000694 effects Effects 0.000 description 16
- 238000012360 testing method Methods 0.000 description 13
- 231100001274 therapeutic index Toxicity 0.000 description 13
- 231100000915 pathological change Toxicity 0.000 description 12
- 230000036285 pathological change Effects 0.000 description 12
- 241000699666 Mus <mouse, genus> Species 0.000 description 10
- 241000709675 Coxsackievirus B3 Species 0.000 description 9
- 230000000840 anti-viral effect Effects 0.000 description 9
- 150000001875 compounds Chemical class 0.000 description 8
- 238000010790 dilution Methods 0.000 description 8
- 239000012895 dilution Substances 0.000 description 8
- 208000015181 infectious disease Diseases 0.000 description 8
- 150000003648 triterpenes Chemical class 0.000 description 8
- 238000003810 ethyl acetate extraction Methods 0.000 description 7
- 230000003612 virological effect Effects 0.000 description 7
- 239000012531 culture fluid Substances 0.000 description 6
- 239000000126 substance Substances 0.000 description 6
- IWUCXVSUMQZMFG-AFCXAGJDSA-N Ribavirin Chemical compound N1=C(C(=O)N)N=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](CO)O1 IWUCXVSUMQZMFG-AFCXAGJDSA-N 0.000 description 5
- 229940079593 drug Drugs 0.000 description 5
- 230000002401 inhibitory effect Effects 0.000 description 5
- 238000002347 injection Methods 0.000 description 5
- 239000007924 injection Substances 0.000 description 5
- 239000002609 medium Substances 0.000 description 5
- 238000011160 research Methods 0.000 description 5
- 239000002356 single layer Substances 0.000 description 5
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 4
- 241001500350 Influenzavirus B Species 0.000 description 4
- 241000699670 Mus sp. Species 0.000 description 4
- 208000036142 Viral infection Diseases 0.000 description 4
- 238000010521 absorption reaction Methods 0.000 description 4
- 238000001514 detection method Methods 0.000 description 4
- 201000010099 disease Diseases 0.000 description 4
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 4
- 238000004519 manufacturing process Methods 0.000 description 4
- 150000007523 nucleic acids Chemical class 0.000 description 4
- 230000009385 viral infection Effects 0.000 description 4
- DIZWSDNSTNAYHK-XGWVBXMLSA-N Betulinic acid Natural products CC(=C)[C@@H]1C[C@H]([C@H]2CC[C@]3(C)[C@H](CC[C@@H]4[C@@]5(C)CC[C@H](O)C(C)(C)[C@@H]5CC[C@@]34C)[C@@H]12)C(=O)O DIZWSDNSTNAYHK-XGWVBXMLSA-N 0.000 description 3
- 239000012980 RPMI-1640 medium Substances 0.000 description 3
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 3
- 230000009471 action Effects 0.000 description 3
- 230000002155 anti-virotic effect Effects 0.000 description 3
- 230000023555 blood coagulation Effects 0.000 description 3
- 238000004364 calculation method Methods 0.000 description 3
- 238000004113 cell culture Methods 0.000 description 3
- 230000002458 infectious effect Effects 0.000 description 3
- 239000004615 ingredient Substances 0.000 description 3
- 108020004707 nucleic acids Proteins 0.000 description 3
- 102000039446 nucleic acids Human genes 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 238000003757 reverse transcription PCR Methods 0.000 description 3
- 238000002791 soaking Methods 0.000 description 3
- 238000001228 spectrum Methods 0.000 description 3
- 230000001629 suppression Effects 0.000 description 3
- 208000011580 syndromic disease Diseases 0.000 description 3
- 229940100050 virazole Drugs 0.000 description 3
- QGJZLNKBHJESQX-UHFFFAOYSA-N 3-Epi-Betulin-Saeure Natural products C1CC(O)C(C)(C)C2CCC3(C)C4(C)CCC5(C(O)=O)CCC(C(=C)C)C5C4CCC3C21C QGJZLNKBHJESQX-UHFFFAOYSA-N 0.000 description 2
- CLOUCVRNYSHRCF-UHFFFAOYSA-N 3beta-Hydroxy-20(29)-Lupen-3,27-oic acid Natural products C1CC(O)C(C)(C)C2CCC3(C)C4(C(O)=O)CCC5(C)CCC(C(=C)C)C5C4CCC3C21C CLOUCVRNYSHRCF-UHFFFAOYSA-N 0.000 description 2
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 2
- 241000991587 Enterovirus C Species 0.000 description 2
- 206010069767 H1N1 influenza Diseases 0.000 description 2
- 241000238631 Hexapoda Species 0.000 description 2
- 241001300737 Maprounea africana Species 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- 238000005481 NMR spectroscopy Methods 0.000 description 2
- 108010019160 Pancreatin Proteins 0.000 description 2
- 240000003085 Quassia amara Species 0.000 description 2
- 235000009694 Quassia amara Nutrition 0.000 description 2
- 241000725643 Respiratory syncytial virus Species 0.000 description 2
- 231100000215 acute (single dose) toxicity testing Toxicity 0.000 description 2
- 238000011047 acute toxicity test Methods 0.000 description 2
- 230000001887 anti-feedant effect Effects 0.000 description 2
- 230000000259 anti-tumor effect Effects 0.000 description 2
- QGJZLNKBHJESQX-FZFNOLFKSA-N betulinic acid Chemical compound C1C[C@H](O)C(C)(C)[C@@H]2CC[C@@]3(C)[C@]4(C)CC[C@@]5(C(O)=O)CC[C@@H](C(=C)C)[C@@H]5[C@H]4CC[C@@H]3[C@]21C QGJZLNKBHJESQX-FZFNOLFKSA-N 0.000 description 2
- 230000004071 biological effect Effects 0.000 description 2
- 210000002798 bone marrow cell Anatomy 0.000 description 2
- 239000013553 cell monolayer Substances 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- 239000000470 constituent Substances 0.000 description 2
- 230000009849 deactivation Effects 0.000 description 2
- PZXJOHSZQAEJFE-UHFFFAOYSA-N dihydrobetulinic acid Natural products C1CC(O)C(C)(C)C2CCC3(C)C4(C)CCC5(C(O)=O)CCC(C(C)C)C5C4CCC3C21C PZXJOHSZQAEJFE-UHFFFAOYSA-N 0.000 description 2
- 238000007865 diluting Methods 0.000 description 2
- 210000003743 erythrocyte Anatomy 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 230000002496 gastric effect Effects 0.000 description 2
- 238000003304 gavage Methods 0.000 description 2
- 230000036541 health Effects 0.000 description 2
- 241000411851 herbal medicine Species 0.000 description 2
- 238000001802 infusion Methods 0.000 description 2
- 238000012423 maintenance Methods 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- MQYXUWHLBZFQQO-UHFFFAOYSA-N nepehinol Natural products C1CC(O)C(C)(C)C2CCC3(C)C4(C)CCC5(C)CCC(C(=C)C)C5C4CCC3C21C MQYXUWHLBZFQQO-UHFFFAOYSA-N 0.000 description 2
- 229940055695 pancreatin Drugs 0.000 description 2
- 231100000614 poison Toxicity 0.000 description 2
- 229940013788 quassia Drugs 0.000 description 2
- 210000002345 respiratory system Anatomy 0.000 description 2
- HZCAHMRRMINHDJ-DBRKOABJSA-N ribavirin Natural products O[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1N1N=CN=C1 HZCAHMRRMINHDJ-DBRKOABJSA-N 0.000 description 2
- 229960000329 ribavirin Drugs 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 210000002784 stomach Anatomy 0.000 description 2
- 201000010740 swine influenza Diseases 0.000 description 2
- 210000001519 tissue Anatomy 0.000 description 2
- 238000004448 titration Methods 0.000 description 2
- 231100000331 toxic Toxicity 0.000 description 2
- 230000002588 toxic effect Effects 0.000 description 2
- -1 triterpene chemical compound Chemical class 0.000 description 2
- 241001529453 unidentified herpesvirus Species 0.000 description 2
- 210000001835 viscera Anatomy 0.000 description 2
- 241000700199 Cavia porcellus Species 0.000 description 1
- 208000031404 Chromosome Aberrations Diseases 0.000 description 1
- 241000709711 Coxsackievirus B5 Species 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- 241000204855 Echovirus E1 Species 0.000 description 1
- 241000709714 Echovirus E11 Species 0.000 description 1
- 241000217450 Echovirus E29 Species 0.000 description 1
- 241000709643 Echovirus E9 Species 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 208000004232 Enteritis Diseases 0.000 description 1
- 206010014909 Enterovirus infection Diseases 0.000 description 1
- 206010061598 Immunodeficiency Diseases 0.000 description 1
- 208000029462 Immunodeficiency disease Diseases 0.000 description 1
- 102100034343 Integrase Human genes 0.000 description 1
- 238000012449 Kunming mouse Methods 0.000 description 1
- 241000158728 Meliaceae Species 0.000 description 1
- 201000009906 Meningitis Diseases 0.000 description 1
- 201000002481 Myositis Diseases 0.000 description 1
- 229930182555 Penicillin Natural products 0.000 description 1
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 1
- 241000709664 Picornaviridae Species 0.000 description 1
- RVMPLOSJMIQORE-NYFXBJJYSA-N Platanic acid Natural products CC(=O)[C@@H]1CC[C@@]2(CC[C@]3(C)[C@@H](CC[C@@H]4[C@@]5(C)CC[C@H](O)C(C)(C)[C@@H]5CC[C@@]34C)[C@@H]12)C(=O)O RVMPLOSJMIQORE-NYFXBJJYSA-N 0.000 description 1
- 206010035664 Pneumonia Diseases 0.000 description 1
- 108010092799 RNA-directed DNA polymerase Proteins 0.000 description 1
- 231100000645 Reed–Muench method Toxicity 0.000 description 1
- 244000097577 Rhus javanica Species 0.000 description 1
- 235000010889 Rhus javanica Nutrition 0.000 description 1
- 241000220317 Rosa Species 0.000 description 1
- 241000607142 Salmonella Species 0.000 description 1
- 229920001617 Vinyon Polymers 0.000 description 1
- 108020000999 Viral RNA Proteins 0.000 description 1
- 241000607479 Yersinia pestis Species 0.000 description 1
- 240000008866 Ziziphus nummularia Species 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 230000001464 adherent effect Effects 0.000 description 1
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
- 229930013930 alkaloid Natural products 0.000 description 1
- 150000003797 alkaloid derivatives Chemical class 0.000 description 1
- DKNWSYNQZKUICI-UHFFFAOYSA-N amantadine Chemical compound C1C(C2)CC3CC2CC1(N)C3 DKNWSYNQZKUICI-UHFFFAOYSA-N 0.000 description 1
- 229960003805 amantadine Drugs 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 230000008485 antagonism Effects 0.000 description 1
- 239000002259 anti human immunodeficiency virus agent Substances 0.000 description 1
- 230000036436 anti-hiv Effects 0.000 description 1
- 238000011951 anti-virus test Methods 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 239000006286 aqueous extract Substances 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- YJEJKUQEXFSVCJ-WRFMNRASSA-N bevirimat Chemical compound C1C[C@H](OC(=O)CC(C)(C)C(O)=O)C(C)(C)[C@@H]2CC[C@@]3(C)[C@]4(C)CC[C@@]5(C(O)=O)CC[C@@H](C(=C)C)[C@@H]5[C@H]4CC[C@@H]3[C@]21C YJEJKUQEXFSVCJ-WRFMNRASSA-N 0.000 description 1
- 230000000975 bioactive effect Effects 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 210000005252 bulbus oculi Anatomy 0.000 description 1
- 230000000747 cardiac effect Effects 0.000 description 1
- 210000004413 cardiac myocyte Anatomy 0.000 description 1
- 210000000748 cardiovascular system Anatomy 0.000 description 1
- 239000012930 cell culture fluid Substances 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 230000007541 cellular toxicity Effects 0.000 description 1
- 231100000005 chromosome aberration Toxicity 0.000 description 1
- 230000015271 coagulation Effects 0.000 description 1
- 238000005345 coagulation Methods 0.000 description 1
- 238000004440 column chromatography Methods 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 210000002249 digestive system Anatomy 0.000 description 1
- 230000000857 drug effect Effects 0.000 description 1
- 238000002651 drug therapy Methods 0.000 description 1
- 238000004043 dyeing Methods 0.000 description 1
- 239000000839 emulsion Substances 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 230000003203 everyday effect Effects 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 229930182478 glucoside Natural products 0.000 description 1
- 150000008131 glucosides Chemical class 0.000 description 1
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 description 1
- 230000035931 haemagglutination Effects 0.000 description 1
- 210000005003 heart tissue Anatomy 0.000 description 1
- 230000007813 immunodeficiency Effects 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 230000002779 inactivation Effects 0.000 description 1
- 230000001524 infective effect Effects 0.000 description 1
- 206010022000 influenza Diseases 0.000 description 1
- 238000002329 infrared spectrum Methods 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 230000000749 insecticidal effect Effects 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 235000015110 jellies Nutrition 0.000 description 1
- 239000008274 jelly Substances 0.000 description 1
- LTHDJUORLSBEHK-UHFFFAOYSA-N kusulactone Natural products C1C(C23C)OC(=O)CC3C(C)(OC(C)=O)C(OC(C)=O)C(O)C2C2(C)C1C(C)C=C(OC)C2=O LTHDJUORLSBEHK-UHFFFAOYSA-N 0.000 description 1
- 125000000686 lactone group Chemical group 0.000 description 1
- 201000004792 malaria Diseases 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 229930014626 natural product Natural products 0.000 description 1
- 210000000653 nervous system Anatomy 0.000 description 1
- 231100000028 nontoxic concentration Toxicity 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 229940049954 penicillin Drugs 0.000 description 1
- 239000000575 pesticide Substances 0.000 description 1
- 150000002989 phenols Chemical class 0.000 description 1
- ULPUGCMMDNWWLH-UHFFFAOYSA-N picraqualide B Natural products COC1=CC(C)C2CC3OC(=O)CC4C(C)(O)C(OC(=O)C)C(OC(=O)C)C(C34C)C2(C)C1=O ULPUGCMMDNWWLH-UHFFFAOYSA-N 0.000 description 1
- RVMPLOSJMIQORE-FUAAEJBOSA-N platanic acid Chemical compound C1C[C@H](O)C(C)(C)[C@@H]2CC[C@@]3(C)[C@]4(C)CC[C@@]5(C(O)=O)CC[C@@H](C(=O)C)[C@@H]5[C@H]4CC[C@@H]3[C@]21C RVMPLOSJMIQORE-FUAAEJBOSA-N 0.000 description 1
- 230000007096 poisonous effect Effects 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- 230000003389 potentiating effect Effects 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 230000001681 protective effect Effects 0.000 description 1
- 206010037844 rash Diseases 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 238000012827 research and development Methods 0.000 description 1
- 230000000630 rising effect Effects 0.000 description 1
- 238000013207 serial dilution Methods 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 208000026425 severe pneumonia Diseases 0.000 description 1
- 239000010865 sewage Substances 0.000 description 1
- 235000015170 shellfish Nutrition 0.000 description 1
- 239000002689 soil Substances 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 239000011550 stock solution Substances 0.000 description 1
- 229940041022 streptomycins Drugs 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 238000002636 symptomatic treatment Methods 0.000 description 1
- 210000001550 testis Anatomy 0.000 description 1
- 238000010257 thawing Methods 0.000 description 1
- 231100000816 toxic dose Toxicity 0.000 description 1
- 239000003440 toxic substance Substances 0.000 description 1
- 231100000820 toxicity test Toxicity 0.000 description 1
- 229960005486 vaccine Drugs 0.000 description 1
- 235000013311 vegetables Nutrition 0.000 description 1
- 239000012873 virucide Substances 0.000 description 1
- 230000029302 virus maturation Effects 0.000 description 1
- 230000002618 waking effect Effects 0.000 description 1
Landscapes
- Medicines Containing Plant Substances (AREA)
Abstract
An extract of brucea fruit for resisting against intestinal virus, influenza virus and adenovirus is prepared from brucea fruit through breaking, immersing in water, heating to 60-100 deg.C, taking supernatant, vacuum concentrating, cyclic extracting in ethyl acetate, vacuum concentrating, filtering by resin column, gradient eluting by the aqueous solution of alcohol, and vacuum concentrating of eluting liquid.
Description
Technical field
The present invention relates to a kind of Chinese medicine extract, extracting method and purposes, particularly relate to a kind of Fructus Bruceae extract, extracting method and purposes.
Background technology
Human life security in many viral disease serious threats, as enterovirus (EV) numerous types, the infection that causes is varied especially, and morbidity is many again to be occurred with syndrome, state of an illness weight great disparity, the infant patient can be broken out severe cardiac myositis or severe pneumonia, and especially neonatal eruption and prevalence can cause death and die.A kind of syndrome can be caused, can be caused different syndromes with a kind of (type) virus by different virus, is the universal phenomenon in the enterovirus genus viral infection.Majority is the performance of subclinical type behind the patient infection enterovirus, is difficult for being found, and it is bigger therefore effectively and in time to make the etiological diagnosis difficulty.Enterovirus be again by excrement-mouth and (or) approach that easily scatters such as respiratory tract infects, should belong to virus acidproof (pH 3.0), ether-resistant simultaneously, still can survive during stomach by the people, survive the long period in a humid environment, can from sewage and mud, be separated to, can detect virus water, soil, vegetable, shellfish, contact contaminated water source, food, marine product, tableware and may cause the propagation of community, often cause sporadic popular or large tracts of land is popular.Except that poliovirus, still not having vaccine can be for prevention at present.Therefore, enterovirus is that current serious influences one of important cause of disease of human health.In addition, the influenza that influenza virus causes, human health in same serious threat such as the pneumonia that adenovirus causes, enteritis, meningitis.
At present, the disease that enterovirus, influenza virus, adenovirus etc. are caused still lacks narrow spectrum specific treatment medicine, the main means that adopt symptomatic treatment, mostly the medicine that is adopted is broad spectrum activity antiviral drugs such as ribavirin, amantadine etc., is badly in need of seeking effective medicine.
Fructus Bruceae is the Chinese medicine of using always, for the mature fruit of quassia (Brucea javanica (L.) Merr.) (" national Chinese herbal medicine compilation " writes group. national Chinese herbal medicine compilation, 1975:596.), call numerous.Its main component is bitter principle compositions such as alkaloid, glucoside, phenols component and picrol, bitter principle, and its main bioactive ingredients is the kusulactone chemical compound, and it belongs to triterpene substance.At present, verified have the triterpene substance of the effect that suppresses virus to have from the isolating pentacyclic triterpene chemical compound of Maprounea africana (Pengsuparp T.Cai L, Fong HH, Kinghorn AD, Pezzuto JM, Wani MC, Wall ME.Pentacyclic triterpenes derived from Maprounea africana are potent inhibitors ofHIV-1 reverse transcriptase.J Nat Prod, 1994,57 (3): 415～418.), from the isolating triterpene of Syzigium daviflorum with fall triterpene (Fujioka T.Kashiwada Y.Kilkuskie RE, Cosentino LM, Ballas LM, Jiang JB, Janzen WP, Chen IS, Lee KH.Anti-AIDS agents, 11.Betulinic acid and platanic acid asanti-HIV principles from Syzigium claviflorum, and the anti-HIV activity of structurally relatedtriterpenoids.J Nat Prod, 1994,57 (2): 243～247.Kanamoto T.Kashiwada Y.Kanbara K, GotohK, Yoshimori M, Goto T.Sano K, Nakashima H.Anti-human immunodeficiency virus activityof YK-FH312 (a betulinic acid derivative), a novel compound blocking viral maturation.Antimicrob Agents Chemother, 2001,45 (4): 1225～1230.) etc., these lactone compounds all have significant inhibitory effect to virus (HIV).Chinese scholar Luo Xiaodong has systematically studied the chemical constituent of multiple Meliaceae pesticide plant and four insect antifeedant activity of falling triterpene and related compound wherein, therefrom isolation identification a chemical compound surplus 200, be noval chemical compound surplus in the of 70 wherein, obtained 5 types 10 surplus individually have an active chemical compound of better insect antifeedant.And the antivirus action of quassia is seldom reported, biological activity in the past focuses mostly at antitumor, malaria, (Luo Xiaodong is gone up in pest-resistant isoreactivity research, Wu Shaohua, Ma Yunbao, Wu Dagang. the insecticidal activity of seal sour jujube extract and wherein four fall triterpene research [J]. research and development of natural products, 2001,13 (1): 9～13. in Rong Min, the few rosy clouds .1985 of woods～quassinoids progress [J] in 1993. Chinese pharmaceutical chemistry magazine, 1994,4 (3): 224～232. fourth rising suns in morning, Suo Yourui. Chinese medicine Fructus Bruceae chemical constituent and pharmaceutical research progress [J]. Chinese patent medicine, 2006,28 (1): 117～120.), and study at most, and its Oleum Fructus Bruceae Emulsion is developed for the antitumor action of Fructus Bruceae.Almost still blank for the research of Fructus Bruceae antivirus action.Fructus Bruceae often is used in the Chinese medicine compound of heat-clearing and toxic substances removing, also Fructus Bruceae is not suppressed the report of viral infection effective ingredient and related compound at present.
Summary of the invention
Of the present inventionly provide a kind of Fructus Bruceae extract.
Second purpose of the present invention provides a kind of extracting method of Fructus Bruceae extract.
The 3rd purpose of the present invention provides a kind of purposes of Fructus Bruceae extract.
Technical scheme of the present invention is summarized as follows:
A kind of Fructus Bruceae extract, make with following method: the Fructus Bruceae of 1 mass parts after (1) will pulverize is 1 in mass ratio: the ratio of 1-3 adds flooding, and each lixiviate 1-3 hour, lixiviate 2-3 time; Add again with the Fructus Bruceae mass ratio be 1: the water of 1-3, be heated to 60--100 ℃ of lixiviate, each lixiviate 1-3 hour, the Fructus Bruceae of 1 mass parts after lixiviate maybe will be pulverized for 2-8 time is 1 in mass ratio: the ratio of 1-3 was soaked 1-3 hour, heating decocts extracts, the each decoction 1-3 hour decocts 1-4 time; (2) the supernatant concentrating under reduced pressure that step (1) is obtained is to the 0.5-2 mass parts, add ethyl acetate and extract repeatedly, to the acetic acid ethyl acetate extract color become colorless transparent till; (3) be evaporated to sepia extractum; (4) be splined on the resin filter post, with concentration of volume percent is that 20%, 40%, 60%, 80%, 100% ethanol water carries out gradient elution, the eluent concentrating under reduced pressure of collecting 40%～80% ethanol water gets brown extractum, is a kind of Fructus Bruceae extract.
The model of described resin is D-101 or D-201 or AB-8 or XAD-7 or HP-10 or HP-20 or HP-21.
Step (1) preferable methods is: the Fructus Bruceae of 1 mass parts after will pulverizing is that 1: 2 ratio adds flooding in mass ratio, each lixiviate 2 hours, lixiviate 3 times; Adding with the Fructus Bruceae mass ratio is 1: 2 water again, is heated to 80 ℃ of lixiviates, each lixiviate 2 hours, lixiviate 7 times.
A kind of extracting method of Fructus Bruceae extract, be to form with following step: the Fructus Bruceae of 1 mass parts after (1) will pulverize is 1 in mass ratio: the ratio of 1-3 adds flooding, and each lixiviate 1-3 hour, lixiviate 2-3 time; Add again with the Fructus Bruceae mass ratio be 1: the water of 1-3, be heated to 60--100 ℃ of lixiviate, each lixiviate 1-3 hour, the Fructus Bruceae of 1 mass parts after lixiviate maybe will be pulverized for 2-8 time is 1 in mass ratio: the ratio of 1-3 was soaked 1-3 hour, heating decocts extracts, the each decoction 1-3 hour decocts 1-4 time; (2) the supernatant concentrating under reduced pressure that step (1) is obtained is to the 0.5-2 mass parts, add ethyl acetate and extract repeatedly, to the acetic acid ethyl acetate extract color become colorless transparent till; (3) be evaporated to sepia extractum; (4) be splined on the resin filter post, with concentration of volume percent is that 20%, 40%, 60%, 80%, 100% ethanol water carries out gradient elution, the eluent concentrating under reduced pressure of collecting 40%～80% ethanol water gets brown extractum, is a kind of Fructus Bruceae extract.
The model of described resin is D-101 or D-201 or AB-8 or XAD-7 or HP-10 or HP-20 or HP-21.
Step (1) preferable methods is: the Fructus Bruceae of 1 mass parts after will pulverizing is that 1: 2 ratio adds flooding in mass ratio, each lixiviate 2 hours, lixiviate 3 times; Adding with the Fructus Bruceae mass ratio is 1: 2 water again, is heated to 80 ℃ of lixiviates, each lixiviate 2 hours, lixiviate 7 times.
The purposes of a kind of Fructus Bruceae extract that makes with said method, the application in the preparation antiviral drugs.Described virus is enterovirus or adenovirus or influenza virus.
Advantage of the present invention:
A kind of Fructus Bruceae extract of the present invention is a kind of triterpene substance that extracts from Fructus Bruceae, and main physicochemical characteristics is: be soluble in dimethyl sulfoxide (DMSO), dissolubility 〉=30%, brown extractum (brown jelly).
Fructus Bruceae extract extracting method of the present invention is the exploitation of the present inventor's independent studies, and particularly two step extractions prepare sample and help the performance of antiviral ingredients effect, and effectively removed its toxic component.
A kind of Fructus Bruceae extract of the present invention has beyond thought antiviral effect, particularly anti-enterovirus, influenza virus and adenovirus.
The specific embodiment
The present invention is further illustrated below in conjunction with specific embodiment.But these embodiment must not be interpreted as and go up limitation of the present invention in all senses.
Embodiment 1
A kind of Fructus Bruceae extract, make with following method: (1) optional homemade high-quality Chinese crude drug Fructus Bruceae, the place of production: major production areas, China south adds water 2kg, room temperature lixiviate, each lixiviate 2 hours, lixiviate 3 times with the Fructus Bruceae of the 1kg after pulverizing; Add water 2kg again, be heated to 80 ℃ of lixiviates, each lixiviate 2 hours, lixiviate 7 times; (2) with the supernatant concentrating under reduced pressure of lixiviate to 1kg, add the ethyl acetate extraction of 1kg, extract repeatedly 5 times, the acetic acid ethyl acetate extract color becomes colorless transparent; (3) collect acetic acid ethyl acetate extract, be evaporated to sepia extractum; (4) ethyl acetate extractum is splined on macroporous resin D-101 Filter column, with concentration of volume percent is that 20%, 40%, 60%, 80%, 100% ethanol water carries out gradient elution, flow velocity keeps 2ml/min, every part effluent volume is 3 times of bed volumes, the eluent concentrating under reduced pressure of collecting 40%～80% ethanol water gets brown extractum, is a kind of Fructus Bruceae extract.
This preparation method mild condition, simple to operate, productive rate is 1.571%, cost is lower.
With reference to State Standard of the People's Republic of China GB15193.3-94, GB15193.5-94, GB15193.8-94, GB15193.7-94, GB15193.4-94, GB15193.14-94 and GB15193.13-94, carry out rat acute toxicity test, acute toxicity test in mice, mouse bone marrow cells micronucleus test (male), mouse bone marrow cells micronucleus test (female), mouse sperm deformity test, mouse testis chromosomal aberration test, Salmonella reversion test, the tertogenicity test of rat system and rat and fed experiment in 30 days, the result shows large and small Mus LD50＞30.0g/kg; The Fructus Bruceae extract (1000mg/kg) of the oral embodiment of the invention of mice 1 preparation, one day 1 time, serve on 15 days, under waking state, observe its spiritual nervous system, cardiovascular system respiratory system of unifying, all no abnormal performance of result.
Embodiment 2
A kind of Fructus Bruceae extract, make with following method: the Fructus Bruceae of the 1kg after (1) will pulverize adds water 1kg, room temperature lixiviate, each lixiviate 3 hours, lixiviate 2 times; Add water 3kg again, be heated to 60 ℃ of lixiviates, each lixiviate 3 hours, lixiviate 2 times; (2) with lixiviate supernatant concentrating under reduced pressure to 0.5kg, add the ethyl acetate extraction of 1kg, extraction repeatedly, to the acetic acid ethyl acetate extract color become colorless transparent till; (3) be evaporated to sepia extractum; (4) be splined on macroporous resin D-201 Filter column, with concentration of volume percent is that 20%, 40%, 60%, 80%, 100% ethanol water carries out gradient elution, flow velocity keeps 2ml/min, every part effluent volume is 3 times of bed volumes, the eluent concentrating under reduced pressure of collecting 40%～80% ethanol water gets brown extractum, is a kind of Fructus Bruceae extract.
Embodiment 3
A kind of Fructus Bruceae extract, make with following method: the Fructus Bruceae of the 1kg after (1) will pulverize adds water 3kg, room temperature lixiviate, each lixiviate 1 hour, lixiviate 3 times; Add water 1kg again, be heated to 100 ℃ of lixiviates, each lixiviate 1 hour, lixiviate 8 times; (2) with the supernatant concentrating under reduced pressure of lixiviate to 2kg, add the ethyl acetate extraction of 1kg, extraction repeatedly, to the acetic acid ethyl acetate extract color become colorless transparent till; (3) be evaporated to sepia extractum; (4) be splined on macroporous resin AB-8 Filter column, with concentration of volume percent is that 20%, 40%, 60%, 80%, 100% ethanol water carries out gradient elution, flow velocity keeps 2ml/min, every part effluent volume is 3 times of bed volumes, the eluent concentrating under reduced pressure of collecting 40%～80% ethanol water gets brown extractum, is a kind of Fructus Bruceae extract.
Embodiment 4
A kind of Fructus Bruceae extract, make with following method: the Fructus Bruceae of the 1kg after (1) will pulverize adds water 1kg, soaking at room temperature 2 hours, heating decocts extracts, and decocts 2 hours at every turn, decocts 3 times; (2) with the supernatant concentrating under reduced pressure that decocts to 1kg, add the ethyl acetate extraction of 1kg, extract repeatedly, to the acetic acid ethyl acetate extract color become colorless transparent till; (3) be evaporated to sepia extractum; (4) be splined on macroporous resin XAD-7 Filter column, with concentration of volume percent is that 20%, 40%, 60%, 80%, 100% ethanol water carries out gradient elution, the eluent concentrating under reduced pressure of collecting 40%～80% ethanol water gets brown extractum, is a kind of Fructus Bruceae extract.
Embodiment 5
A kind of Fructus Bruceae extract, make with following method: the Fructus Bruceae of the 1kg after (1) will pulverize adds water 3kg, soaking at room temperature 1 hour, heating decocts extracts, and decocts 1 hour at every turn, decocts 4 times; (2) with the supernatant concentrating under reduced pressure that decocts to 0.5kg, add the ethyl acetate extraction of 1kg, extract repeatedly, to the acetic acid ethyl acetate extract color become colorless transparent till; (3) be evaporated to sepia extractum; (4) be splined on macroporous resin HP-10 Filter column, with concentration of volume percent is that 20%, 40%, 60%, 80%, 100% ethanol water carries out gradient elution, the eluent concentrating under reduced pressure of collecting 40%～80% ethanol water gets brown extractum, is a kind of Fructus Bruceae extract.
Embodiment 6
A kind of Fructus Bruceae extract, make with following method: the Fructus Bruceae of the 1kg after (1) will pulverize adds water 2kg, soaking at room temperature 3 hours, heating decocts extracts, and decocts 3 hours at every turn, decocts 1 time; (2) with the supernatant concentrating under reduced pressure that decocts to 2kg, add the ethyl acetate extraction of 1kg, extract repeatedly, to the acetic acid ethyl acetate extract color become colorless transparent till; (3) be evaporated to sepia extractum; (4) be splined on macroporous resin HP-20 (also can select HP-21 for use) Filter column, with concentration of volume percent is that 20%, 40%, 60%, 80%, 100% ethanol water carries out gradient elution, the eluent concentrating under reduced pressure of collecting 40%～80% ethanol water gets brown extractum, is a kind of Fructus Bruceae extract.
Embodiment 7
The anti-enterovirus effect of Fructus Bruceae extract
Fructus Bruceae extract has anti-preferably enterovirus effect in vivo, outward.
One, the external anti-enterovirus effect research of Fructus Bruceae extract
1, Fructus Bruceae extract in Vero E6 cell culture to the inhibitory action of enterovirus
(1) cell culture: the culture bottle that covers with Vero E6 cell adds 0.25% pancreatin (preparation of Hanks liquid), and 37 ℃ digested 5 minutes, and added the RPMI-1640 culture fluid and (contain hyclone 10%, 3% glutamine 1%) piping and druming is gone down to posterity at 1: 3, and 2-3d covers with, add the cell counting count board counting, be configured to 10
5Cells/ml, inoculating cell culture plate, the every hole 0.2ml of 96 orifice plates, the every hole 1ml of 24 orifice plates, 37 ℃, 5%CO
2Cultivate 24h, cell experimentizes after growing up to monolayer.
(2) medicine pair cell toxicity test: after will the Vero E6 cell of enterovirus susceptible not being added Versene Digestive system 4ml digestion with 0.25% pancreatin 1ml, with growth-promoting media blow off, mixing, 100 μ l/ holes are inoculated in 96 porocyte culture plates, 37 ℃, 5%CO
2Grow into cell monolayer behind middle cultivation 24h～48h, discard culture fluid, stand-by.Dilute by variable concentrations keeping liquid with RPMI-1640 behind the given the test agent autoclaving, do 6 concentration of 2 times of serial dilutions and carry out scalping.Each concentration inoculation preprepared cell 3 hole, every hole 100 μ l.With positive control drug ribavirin injection (not filtering), by different concentration dilutions, every concentration 4 holes, every hole 100 μ l.Establish normal cell Vero E6 simultaneously, 37 ℃, 5%CO
2The middle cultivation.Rose in 1st, observation of cell form day by day under inverted microscope, with every hole less than 25%, pathological changes is+; 25%～50% is ++; 50%～75% is +++; 75%～100% is ++ ++ (with pathological changes ++ ++ be terminal point, be 4d observing time).Calculate maximal non-toxic concentration C C according to Reed Muench method
0With the poisonous concentration C C of half
50
(3) Fructus Bruceae extract inhibitory action that enterovirus is duplicated: inoculating cell Vero E6 inhales and removes culture fluid, with 100 * TCID after 96 orifice plates grow up to 70%～80% monolayer
50Virus 100 μ l absorption Vero E6 cell is hatched 2h for 37 ℃, and the flush away free virus adds with the Fructus Bruceae sample liquid 100 μ l that keep the liquid doubling dilution, 37 ℃, 5%CO
2Continue in the incubator to cultivate, the observation of cell pathological changes, record pathological changes hole count, calculation sample calculates medium effective concentration (EC to the suppression ratio of virus
50) and therapeutic index (TI).
(4) Fructus Bruceae extract is to the inhibitory action of enterovirus adherent cell: inoculating cell is inhaled and is removed culture fluid, 100 * TCID after 96 orifice plates grow up to 70%～80% monolayer
50Virus 100 μ l and 100 μ l 59
#The sample sample diluting liquid adds (volume ratio is 1: 1) simultaneously, hatches 2h for 37 ℃, removes liquid, adds 100 μ l and keeps liquid, 37 ℃, 5%CO
2Continue to cultivate 96h in the incubator, the observation of cell pathological changes, record pathological changes hole count, calculation sample calculates medium effective concentration (EC to the suppression ratio of virus
50) and therapeutic index (TI).
(5) the Fructus Bruceae extract pretreatment cell is to the retardation (protective effect) of enterovirus infection: inoculating cell is after 96 orifice plates grow up to 70%～80% monolayer; culture fluid is removed in suction, adds 100 μ l sample diluting liquids, and cell and sample are hatched 2h for 37 ℃; discard sample liquid, add 100 * TCID
50Virus liquid 100 μ l are hatched 2h for 37 ℃, and the flush away free virus adds and keeps liquid and hatch 200 μ l, 37 ℃, 5%CO
2Continue to cultivate 96h in the incubator, the observation of cell pathological changes, record pathological changes hole count, calculation sample calculates medium effective concentration (EC to the suppression ratio of virus
50) and therapeutic index (TI).
(6) Fructus Bruceae extract is to the deactivation of enterovirus: the Fructus Bruceae extract of getting embodiment 1 preparation of 50 μ g/ml and 30 μ g/ml is hatched 2h with virus stock solution used in 37 ℃ respectively, make 10 times of gradient dilutions with the hybrid virus liquid after keeping liquid and will acting on, be inoculated in 96 well culture plates that cover with cell monolayer and carry out titration of virus, each dilution factor repeats 4 holes, viral liquid with equal extension rate is done contrast, 37 ℃, 5%CO
2Incubator continues to cultivate 96h, and the observation of cell pathological changes is calculated virus titer and the sample inactivation ratio to virus.
2, experimental result
(1) Fructus Bruceae extract is to the inhibitory action of duplicating of 7 kinds of enterovirus.
| Virus | Fructus Bruceae extract | Virazole | ||
| EC 50(μg/ml) | TI | EC 50(μg/ml) | TI | |
| Coxsackie virus B3 Coxsackie virus B5 Echo virus 1 Echo virus 9 Echo virus 11 Echo virus 29 Polio virus I | 2.00 5.00 2.67 2.50 3.45 1.75 7.50 | 31.5 12.6 23.6 25.2 18.3 36.0 8.4 | ≥250.0 ≥250.0 - ≥125.6 - - ≥500.0 | 4 4 - 8 - - 2 |
(2) Fructus Bruceae extract can not suppress the adsorption of virus.
(3) Fructus Bruceae extract has stronger deactivation enterovirus effect, can make the 7 kinds of virus titers of testing reduce by 2～4 orders of magnitude.
Two, the anti-non-enterovirus effect of Fructus Bruceae extract
1, inoculating cell is cultured to 70%～80% monolayer in 96 porocyte plates, abandons culture fluid, uses Hank ' s liquid to wash 1 time, adds to be diluted to 100 * TCID
50Viral liquid, every hole 100 μ l, 35 ℃ absorption 2h.Sample liquid is begun to do doubling dilution totally 8 concentration from the half toxic dose.Discard viral liquid after the absorption, use Hank ' s liquid to wash 1 time, add the good medicinal liquid of dilution, each concentration adds 4 holes, establishes the contrast of virus control and cell simultaneously, cultivates 5d for 37 ℃, and observation had or not CPE and write down the result every day.The terminal point determining of the drug dilution degree of pathological changes (++) as this medicine minimum effective drug concentration takes place with cell 50%, more than three test triplicate at least, all write down the result at every turn.
2, the resisiting influenza virus effect then need with-20 ℃ of culture plate incomes, be carried out the experiment of blood clotting titre to each experimental group after finishing above CPE observation
[98], judge the minimum effective drug concentration of medicine to this influenza virus: get vinyon plate at the bottom of 96 hole circles, queue every hole from second and add 50 μ l normal saline, the every hole of first row adds medicine 100 μ l to be checked, takes out 50 μ l in first round to do doubling dilution backward; Add 1% guinea-pig red blood cell, 100 μ l, put observed result behind room temperature 30～60min.With erythrocyte 50% coagulation (++) is the final decision result.
3, result of the test
(1) Fructus Bruceae extract of embodiment 1 preparation differs greatly to 6 kinds of non-picornavirus antiviral effects, and is best to the inhibition effect of Adenovirus serotype III, EC
50Be 3.29 μ g/ml, TI is 19.1.Effect to Herpes virus I, Influenza virus A1, Influenza virus A3, Influenza virus B, Respiratory syncytial virus is relatively poor, therefore its TI can think that all less than 4 the Fructus Bruceae extract is to the no antivirus action of other virus.
Fructus Bruceae is got the drug effect of thing and the external anti-non-enterovirus of virazole
| Virus Virus | Fructus Bruceae extract | Virazole | ||
| EC 50(μg/ml) | TI | EC 50(μg/ml) | TI | |
| Herpes virus I Adenovirus serotype III Influenza virus A1 H1N1 Influenza virus A3 H3N2 Influenza virus B Respiratory syncytial virus | 16.74 3.29 17.67 15.43 18.84 18.26 | 3.8 19.1 1.8 2.1 1.7 3.5 | 20.37 10.45 3.99 4.02 13.93 6.97 | 12.3 23.9 15.7 15.6 17.9 30.1 |
(2) with the Fructus Bruceae extract of 50 μ g/ml and 30 μ g/ml respectively at 35 ℃ with influenza virus Influenza virus A1 (H1N1), Influenza virus A3 (H3N2), Influenza virus B effect 2h after, the mixed liquor blood clotting titre viral with contrast after the difference detection effect, as shown in the table: the Fructus Bruceae extract of 50 μ g/ml and 30 μ g/ml all can effectively reduce the blood clotting titre of influenza virus.
Fructus Bruceae extract is to the influence of hirst's hemagglutination titre
The virucide results of the aqueous extract to Influenza virus by the HA test
| Influenza virus | Contrast | 30.0μg/ml | 50.0μg/ml |
| Influenza virus A1 H1N1 Influenza virus A3 H3N2 Influenza virus B | 1∶128 1∶64 1∶128 | 1∶8 1∶4 1∶4 | 1∶8 1∶4 1∶4 |
Three, anti-Coxsackie virus B3 effect in the Fructus Bruceae extract body
1, mice Coxsackie virusB3 viral infection: 50 of kunming mices, being divided into is 5 groups, 10 every group, male and female half and half.After each organizes injected in mice position routine disinfection, (except that A group normal control group) lumbar injection Coxsackie virusB3 virus liquid 0.1ml (10
5* TCID
50), carry out the gastric infusion first time behind the injection 2h.
2, Drug therapy test: carry out the gastric infusion first time behind the 2h behind the lumbar injection Coxsackie virus B3 virus liquid.The A group is the normal control group, and the cell maintenance medium 0.1ml of injection Isodose gavages normal saline during infective virus when irritating stomach; The B group gavages normal saline for the viral infection matched group; The C group is antiviral Fructus Bruceae extract low dosage (1mg/ml) treatment group; The D group is dosage (10mg/ml) treatment group in the antiviral Fructus Bruceae extract; The E group is antiviral Fructus Bruceae extract high dose (100mg/ml) treatment group.Once a day, each 0.2ml.Disease symptom behind the observation zoogenetic infection.Each treated animal is respectively put to death half respectively at 5d and 10d, get blood system from serum behind the excision eyeball, and animal hearts is got by the sterile working.1/2 of each heart sample is used for infectious virus and separates, the detection that is used for Coxsackie virusB3 viral nucleic acid of residue heart sample 1/2.
3, detection method
(1) separation, the evaluation of infectious Coxsackie virus B3 virus in the heart: get mouse heart 1/2 homogenate that above-mentioned Coxsackie virus B3 infects back 5d and 10d, make suspension with the RPMI-1640 cell culture fluid that contains 100U/ml penicillin, 100 μ g/ml streptomycins, get 0.1ml and infect Vero E6 cell, 37 ℃, 5%CO
2Absorption 2h (every the 30min vibration once) abandons supernatant, adds cell maintenance medium, 37 ℃, 5%CO
2Incubator is cultivated.Observe down continuously 6d in inverted microscope, it is positive CPE person to occur, carries out titration of virus after the freeze thawing 3 times, and adopts RT-PCR to identify.CPE person not occurring does not still have CPE person through 3 generations of blind passage and is considered as separating negative, abandons it.
(2) Coxsackie virus B3 viral nucleic acid detects in the heart tissue: after getting mouse heart 1/2 homogenate of above-mentioned Coxsackie virus B3 infection back 5d and 10d, extracting total tissue RNA and viral RNA wherein, with method extracting Coxsackie virus B3, carry out the RT-PCR amplification respectively, product is through agarose gel electrophoresis, EB dyeing, uviol lamp is observed down, and with the contrast of molecular weight standard product, the person is positive band to the occur at the 300bp place.
4, experimental result
Respectively put to death half respectively at 5d and 10d, mouse heart is got by the sterile working, and 1/2 of each heart sample is used for infectious virus and separates, and 1/2 internal organs are used for the detection of Coxsackie virus B3 viral nucleic acid.Vero E6 cell separation virus result shows, is inoculated in respectively organizing mouse tissue virus extracting solution and all can not making cell generation pathological changes of culture plate, and with 3 generations of cell blind passage, cell growth state is good, no pathological changes generation.
And 1/2 internal organs of other sample are extracted total RNA, detect the specific nucleic acid fragment of Coxsackie virus B3 by RT-PCR, the result shows, virus control group, middle dosage group and low dose group result are positive, blank group, high dose group result are negative, under high concentration (100mg/ml), the Fructus Bruceae extract sample can suppress infection and the breeding of Coxsackie virusB3 in the mouse cardiac muscle cell.
Carried out antivirus test by extract to Fructus Bruceae extract sample, different batches Fructus Bruceae extract sample and the Different Extraction Method in source, the different places of production, the result shows: source, the different places of production and different extraction batch do not have obvious influence to the extract antiviral activity, and that extracting method enantiopathy toxic effect really influences is bigger, wherein best with the extraction effect of two-step method (room temperature lixiviate and 80 ℃ of lixiviates combine) antagonism virus composition.
Employing act as the biological activity tracking with anti-Coxsackie virus B3 Fructus Bruceae antiviral component is separated, obtain antiviral active components through flooding, ethyl acetate extraction, macroporous resin column chromatography ethanol elution respectively, through the test of chemical property systematicness, nuclear magnetic resonance, NMR
1The H spectrum,
13C spectrum, Fourier infrared spectrum Preliminary Identification, the anti-Coxsackievirus B3 of Fructus Bruceae active component is lactone materials such as C, D, its overall recovery is that 15.71 ‰, TI are 33.07.
Claims (10)
1. Fructus Bruceae extract, it is characterized in that making with following method: the Fructus Bruceae of 1 mass parts after (1) will pulverize is 1 in mass ratio: the ratio of 1-3 adds flooding, and each lixiviate 1-3 hour, lixiviate 2-3 time; Add again with the Fructus Bruceae mass ratio be 1: the water of 1-3, be heated to 60--100 ℃ of lixiviate, each lixiviate 1-3 hour, the Fructus Bruceae of 1 mass parts after lixiviate maybe will be pulverized for 2-8 time is 1 in mass ratio: the ratio of 1-3 was soaked 1-3 hour, heating decocts extracts, the each decoction 1-3 hour decocts 1-4 time; (2) the supernatant concentrating under reduced pressure that step (1) is obtained is to the 0.5-2 mass parts, add ethyl acetate and extract repeatedly, to the acetic acid ethyl acetate extract color become colorless transparent till; (3) be evaporated to sepia extractum; (4) be splined on the resin filter post, with concentration of volume percent is that 20%, 40%, 60%, 80%, 100% ethanol water carries out gradient elution, the eluent concentrating under reduced pressure of collecting 40%～80% ethanol water gets brown extractum, is a kind of Fructus Bruceae extract.
2. a kind of Fructus Bruceae extract according to claim 1, the model that it is characterized in that described resin are D-101 or D-201 or AB-8 or XAD-7 or HP-10 or HP-20 or HP-21.
3. a kind of Fructus Bruceae extract according to claim 1 and 2 is characterized in that step (1) is that 1: 2 ratio adds flooding for the Fructus Bruceae of 1 mass parts after will pulverizing in mass ratio, and lixiviate is 2 hours at every turn, lixiviate 3 times; Adding with the Fructus Bruceae mass ratio is 1: 2 water again, is heated to 80 ℃ of lixiviates, each lixiviate 2 hours, lixiviate 7 times.
4. the extracting method of a Fructus Bruceae extract, it is characterized in that forming with following step: the Fructus Bruceae of 1 mass parts after (1) will pulverize is 1 in mass ratio: the ratio of 1-3 adds flooding, and lixiviate 1-3 hour at every turn, lixiviate 2-3 time; Add again with the Fructus Bruceae mass ratio be 1: the water of 1-3, be heated to 60--100 ℃ of lixiviate, each lixiviate 1-3 hour, the Fructus Bruceae of 1 mass parts after lixiviate maybe will be pulverized for 2-8 time is 1 in mass ratio: the ratio of 1-3 was soaked 1-3 hour, heating decocts extracts, the each decoction 1-3 hour decocts 1-4 time; (2) the supernatant concentrating under reduced pressure that step (1) is obtained is to the 0.5-2 mass parts, add ethyl acetate and extract repeatedly, to the acetic acid ethyl acetate extract color become colorless transparent till; (3) be evaporated to sepia extractum; (4) be splined on the resin filter post, with concentration of volume percent is that 20%, 40%, 60%, 80%, 100% ethanol water carries out gradient elution, the eluent concentrating under reduced pressure of collecting 40%～80% ethanol water gets brown extractum, is a kind of Fructus Bruceae extract.
5. the extracting method of a kind of Fructus Bruceae extract according to claim 4, the model that it is characterized in that described resin are D-101 or D-201 or AB-8 or XAD-7 or HP-10 or HP-20 or HP-21.
6. according to claim 4 or 5 described a kind of Fructus Bruceae extracts, it is characterized in that step (1) is that 1: 2 ratio adds flooding for the Fructus Bruceae of 1 mass parts after will pulverizing in mass ratio, lixiviate is 2 hours at every turn, lixiviate 3 times; Adding with the Fructus Bruceae mass ratio is 1: 2 water again, is heated to 80 ℃ of lixiviates, each lixiviate 2 hours, lixiviate 7 times.
7. the purposes of the described a kind of Fructus Bruceae extract of one of claim 1-3 is characterized in that the application in the preparation antiviral drugs.
8. according to the purposes of the described a kind of Fructus Bruceae extract of claim 7, it is characterized in that described virus is enterovirus.
9. according to the purposes of the described a kind of Fructus Bruceae extract of claim 7, it is characterized in that described virus is adenovirus.
10. according to the purposes of the described a kind of Fructus Bruceae extract of claim 7, it is characterized in that described virus is influenza virus.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200610014349 CN1895300A (en) | 2006-06-14 | 2006-06-14 | Kosam extract, its extraction and use |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200610014349 CN1895300A (en) | 2006-06-14 | 2006-06-14 | Kosam extract, its extraction and use |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN1895300A true CN1895300A (en) | 2007-01-17 |
Family
ID=37608038
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 200610014349 Pending CN1895300A (en) | 2006-06-14 | 2006-06-14 | Kosam extract, its extraction and use |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN1895300A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102188459A (en) * | 2010-03-09 | 2011-09-21 | 胡清文 | Brucea javanica total terpenoid extractive, and preparation method and application thereof |
| CN102924467A (en) * | 2012-11-07 | 2013-02-13 | 福建农林大学 | A preparation method for extracting and purifying brucein D from Brucea javanica |
| CN113616717A (en) * | 2021-08-19 | 2021-11-09 | 北京华夏百草健康医学研究院(有限合伙) | Pure plant compound liquid for removing wart and nevus and improving skin layer |
-
2006
- 2006-06-14 CN CN 200610014349 patent/CN1895300A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102188459A (en) * | 2010-03-09 | 2011-09-21 | 胡清文 | Brucea javanica total terpenoid extractive, and preparation method and application thereof |
| CN102924467A (en) * | 2012-11-07 | 2013-02-13 | 福建农林大学 | A preparation method for extracting and purifying brucein D from Brucea javanica |
| CN102924467B (en) * | 2012-11-07 | 2015-04-01 | 福建农林大学 | A preparation method for extracting and purifying brucein D from Brucea javanica |
| CN113616717A (en) * | 2021-08-19 | 2021-11-09 | 北京华夏百草健康医学研究院(有限合伙) | Pure plant compound liquid for removing wart and nevus and improving skin layer |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102114044B (en) | Artificially processed bear bile powder and preparation method thereof | |
| WO2017133468A1 (en) | Pulchinenoside and application as inhibitor of ev71 virus | |
| CN112220799A (en) | A product and use for inhibiting virus | |
| CN102895326B (en) | Traditional Chinese medicine composition for treating infantile common cold and preparation method for traditional Chinese medicine composition | |
| CN101875648B (en) | Method for extracting and purifying Pinostrobin from medicinal plants, and pharmaceutical preparation and application thereof | |
| CN107136370A (en) | A kind of herb fermenting beverage with antitumor efficacy and preparation method thereof | |
| CN1895300A (en) | Kosam extract, its extraction and use | |
| CN1330330C (en) | Antivirus medicinal composition, preparation method and use | |
| CN111494436A (en) | Anti-hypoxia application of chaenomeles speciosa mycelium and extract thereof | |
| CN113181229B (en) | Application of cabbage type rape-isatis tinctoria G monomer addition system in inhibiting novel coronavirus SARS-CoV-2 | |
| CN101474247A (en) | Application of spatholobus stem extract in preparing medicament for resisting influenza virus and enterovirus | |
| CN1118281C (en) | Application of seeweed polyose sulphate | |
| CN113797272A (en) | Chinese herbal medicine compound preparation for preventing and treating prawn vibrio parahaemolyticus disease and preparation method thereof | |
| CN109288902B (en) | Preparation method and application of total flavonoid fermentation product of jujube leaves with strong antioxidant activity | |
| CN101084991B (en) | A kind of Tibetan medicine Tangut green orchid extract and its application in the preparation of antiviral drugs | |
| CN106177035B (en) | Preparation method and application of effective rosa chinensis flower extract with blood sugar reducing and anticancer functions | |
| CN110898086B (en) | Composite antiviral preparation of black tiger palm extract and preparation method and application thereof | |
| CN119970810B (en) | Antiviral and antibacterial composition and preparation method and application thereof | |
| CN103816199B (en) | A kind of anti-duck virus hepatitis Chinese medical extract compound | |
| CN1233358C (en) | Method for extractings ubstance resisting respiratory syncytial virus from patrinia | |
| CN116036187B (en) | A compound traditional Chinese medicine for preventing coccidiosis in chickens and its preparation method and application | |
| CN114209732B (en) | Medicine for preventing and treating nocardiosis and application thereof | |
| CN112957388B (en) | Application of brassica napus-isatis tinctoria E monomer addition system in inhibiting influenza virus | |
| CN110141590B (en) | Compound composition of seaside mallow traditional Chinese medicine for preventing chicken coccidiosis and its application | |
| CN101461820A (en) | Application of mangiferin in preparing anti-influenza virus medicament |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C02 | Deemed withdrawal of patent application after publication (patent law 2001) | ||
| WD01 | Invention patent application deemed withdrawn after publication |