CN105399669B - A kind of dihydroxyisoquinoline and its preparation method and application from centipede - Google Patents
A kind of dihydroxyisoquinoline and its preparation method and application from centipede Download PDFInfo
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- CN105399669B CN105399669B CN201510847846.3A CN201510847846A CN105399669B CN 105399669 B CN105399669 B CN 105399669B CN 201510847846 A CN201510847846 A CN 201510847846A CN 105399669 B CN105399669 B CN 105399669B
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- 238000011081 inoculation Methods 0.000 description 1
- 229910052742 iron Inorganic materials 0.000 description 1
- VYCKDIRCVDCQAE-UHFFFAOYSA-N isoquinolin-3-amine Chemical class C1=CC=C2C=NC(N)=CC2=C1 VYCKDIRCVDCQAE-UHFFFAOYSA-N 0.000 description 1
- 229930013397 isoquinoline alkaloid Natural products 0.000 description 1
- 238000011031 large-scale manufacturing process Methods 0.000 description 1
- 230000031700 light absorption Effects 0.000 description 1
- 201000007227 lymph node tuberculosis Diseases 0.000 description 1
- 230000003211 malignant effect Effects 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 230000009401 metastasis Effects 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 239000012452 mother liquor Substances 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 230000035764 nutrition Effects 0.000 description 1
- 229960003104 ornithine Drugs 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 235000010603 pastilles Nutrition 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 238000005191 phase separation Methods 0.000 description 1
- COLNVLDHVKWLRT-UHFFFAOYSA-N phenylalanine Natural products OC(=O)C(N)CC1=CC=CC=C1 COLNVLDHVKWLRT-UHFFFAOYSA-N 0.000 description 1
- 231100000614 poison Toxicity 0.000 description 1
- 239000002574 poison Substances 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 235000020995 raw meat Nutrition 0.000 description 1
- 206010039073 rheumatoid arthritis Diseases 0.000 description 1
- 210000001563 schizont Anatomy 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 239000002002 slurry Substances 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 238000010183 spectrum analysis Methods 0.000 description 1
- 230000001629 suppression Effects 0.000 description 1
- 208000011580 syndromic disease Diseases 0.000 description 1
- 229960003080 taurine Drugs 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 229940126680 traditional chinese medicines Drugs 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 210000003812 trophozoite Anatomy 0.000 description 1
- 239000012588 trypsin Substances 0.000 description 1
- JFJZZMVDLULRGK-URLMMPGGSA-O tubocurarine Chemical compound C([C@H]1[N+](C)(C)CCC=2C=C(C(=C(OC3=CC=C(C=C3)C[C@H]3C=4C=C(C(=CC=4CCN3C)OC)O3)C=21)O)OC)C1=CC=C(O)C3=C1 JFJZZMVDLULRGK-URLMMPGGSA-O 0.000 description 1
- 239000004474 valine Substances 0.000 description 1
- 231100000611 venom Toxicity 0.000 description 1
- 238000003809 water extraction Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D217/00—Heterocyclic compounds containing isoquinoline or hydrogenated isoquinoline ring systems
- C07D217/22—Heterocyclic compounds containing isoquinoline or hydrogenated isoquinoline ring systems with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to carbon atoms of the nitrogen-containing ring
- C07D217/24—Oxygen atoms
-
- 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
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention discloses a kind of dihydroxy isoquinolin and its methods and applications prepared from centipede, belong to drug field.Dihydroxy isoquinolin in the present invention is 3,8- dihydroxy isoquinolin, extracts and obtains from centipede, can be used for treating the treatment of malaria, sterilization and entity tumor.The malaria is to parasitize the Plasmodium vivax of human body, malariae, plasmodium falciparum and Plasmodium ovale；The bacterium is Escherichia coli, staphylococcus aureus, pseudomonas aeruginosa, first/beta hemolytic streptococcus, streptococcus pneumonia and bloodthirsty Bacillus influenzae；The tumour is incidence, brain, thyroid gland, pancreas, lungs, liver, oesophagus, stomach, mammary gland, kidney, gall-bladder, colon or rectum, ovary, uterus, cervix, the primary or secondary cancer and sarcoma of prostate, bladder, testis originating from people.Toxic side effect of the present invention is smaller, can be used for the treatment and adjuvant treatment of various cancers.
Description
Technical field
The present invention relates to drug fields, and in particular to extract a kind of noval chemical compound 3 from centipede, 8- dihydroxy isoquinolin with
And application of the active material in anti-malarial activity, antibacterial activity and anti-tumor activity medicine.
Background technique
Malignant tumour is the primary killer of human health.Although traditional tumour treatment has certain curative effect, due to chemistry
The non-specific cytotoxicity of drug has biggish toxic side effect to patient, while being also easy to generate drug resistance, gives tumor patient
Bring great pain.Since proposing tumor vessel hyperplasia theory from Folkman in 1972, by inhibit tumor vessel hyperplasia come
The report for treating tumour emerges in multitude, and achieves immense success.Tumor Angiongesis is the morphology of growth and metastasis of tumours
Basis.Most malignant entity tumors such as oophoroma, liver cancer, cervical carcinoma and breast cancer etc. are all blood vessel dependent tumors.Newly
On the one hand angiogenic provides nutrition and oxygen, the important channel of another aspect or metastases for tumour growth.Therefore, inhibit
Tumor neogenetic blood vessels can effectively inhibit the generation, development and transfer of tumour.
Centipede is terrestrial arthropod, all totally 22 links.Head kermesinus or bronzing, slightly gloss, there is head plate
Covering, head plate subcircular, front end is slightly prominent, and gnathopodite a pair is posted in two sides, and front end two sides have feeler a pair of.The first backboard of trunk
Homochromy with head plate, remaining 20 backboard is brown-green or blackish green, has gloss, often has two from the 4th backboard to the 20th backboard
Longitudinal furrow lines；Abdomen is faint yellow or brown color, shrinkage；From the second section, every section two sides have step a pair of；Step yellow is reddish brown
Color occasionally has yellow-white, is in hooked, the last is to step shape of tail, therefore also known as uropodium, easy to fall off.Matter is crisp, and section has crack.Gas
Micro- raw meat, there is a special pungent foul smell, acrid flavour, micro- salty.Major function relieve the wind syndrome antispastic, function poison dissipating bind, removes obstruction in channels to relieve pain.It is frightened for children
Wind, spasm of twitching, middle air port is askew, hemiplegia, tetanus, rheumatoid arthritis stubborn, sore, scrofula, venomous snake bite.
Complicated component in centipede, the toxic component containing two kinds of similar bee venom, i.e. histamine sample substance and hemolytic protein；Still
Fatty oil, cholesterine, formic acid etc..δ-oxylysine was once isolated again；Amino acid has histidine, arginine, ornithine, relies
Propylhomoserin, glycine, alanine, valine, leucine, phenylalanine, serine, taurine, glutamic acid.Through Literature Consult, have
(the practical oncology of Primary Study [J] of the centipedes tissue extract anti-tumor activity such as Qu Aibing, Zhao Weicheng, Liang Liang is miscellaneous by researcher
Will, 2003,17 (1): 29-30) centipede tissue extract detected using mtt assay, find its to stomach cancer cell and liver cancer cells compared with
Strong cytotoxicity.There are also researchers (in Wang Shuo, Qin Wenhui Scolopndra subspinipes multidens (Newport) and Scolopendra subspinipes antitumor action research [J]
Pharmacology of traditional Chinese medical formulae magazine is tested by state, 2011,13:156-158) the even of the huge centipede of more spine and Scolopendpa Subspinipes Mutilans L. KOCH is detected using identical method
Effect of the slurries to Human hepatoma cell line Bel-7402, confirming two kinds of centipedes has stronger antitumor action.Liberation army the 4th
Five or eight hospitals are (when Han Li, Zhou Yongqin, Han Yu Scolopendra extract induce cervical cancer Hela cells apoptosis and its research [J] of mechanism
Precious traditional Chinese medical science traditional Chinese medicines, 2007,18 (9): 2109-2111) influence that centipede is proliferated cervical cancer Hela cells is observed in a similar fashion,
Prove that the ether of centipede, ethanol extract have significant anti tumor activity in vitro to Hela cell.Nanjing Normal University Jiangsu
Province molecular medicine biotechnology key lab is (in antibacterial activity [J] of the Scolopendra subspinipes water extract such as Ren Wenhua, Zhang Shuanquan
Medicinal material, 2007,01:10-14) it is extracted centipede water extract and determines it with certain antibacterial activity.But at present there has been no
Research has shown that there is which kind of substance is antitumor or antibacterial activity component be specifically in centipede, since the component of Scolopendra extract is multiple
It is miscellaneous, and may be also containing the toxic component similar to bee venom, therefore centipede is preparing answering in antitumor or antibacterials field
With being constantly subjected to limit.
It is mostly isoquinoline alkaloid in antineoplastic alkaloids, which is widely used, and structure has multiplicity
Property.Rexahn company (Lee Young Bok, Ahn Chang-Ho, Cho Won-Jea.Substituted 3- (hetero)
arylisquinolinamine derivatives for therapeutic applications.WO 2008063548
A2,2008.5.29) possess 5,6 or 7 substitution 3- aminoisoquinoline derivatives can produce antitumor action, can significantly press down
Make the growth of resistance to taxol (taxol) HCT-15 human colon cancer cell model of nude mice bearing tumor tumour.Chinese Patent Application No. is
201210098631.2, data of publication of application is that the patent application document on July 25th, 2012 discloses dihydroisoquinoliness chemical combination
Object and its as the application for preparing plants antimicrobial drug has synthesized a series of dihydroisoquinoliness compounds as planting in the invention
Object antibacterials have good inhibiting effect to various plants pathogen.Chinese Patent Application No. is 201280041436.4,
Data of publication of application is that the patent application document on July 16th, 2014 discloses a kind of substituted 2- alkyl -1- oxo-N-phenyl -
3- heteroaryl -1,2,3,4- tetrahydroisoquinoline -4- amides, can be used for preventing and treating malaria.Although at present about iloquinoline derivative
There are many research of the compound for antibacterial or antineoplaston, and the compound about other structures is used for antibacterial or antineoplaston
Research it is also very much, but it is always unknown in field which kind of specific component materials, which have antitumor and antibacterial functions, in centipede
, so determining the specific structure of the active ingredients substances in centipede has very important medical value.
Summary of the invention
1. to solve the problems, such as
For centipede in the prior art active component it is indefinite, using being restricted the problems such as, the present invention provides one kind
Dihydroxy isoquinolin and its methods and applications prepared from centipede, the active component that the present invention is extracted from centipede are 3,8- bis-
Isoquinolinol has the function of anti-malarial, antibacterial and antitumor, the specific structure of active component in centipede has been determined for the first time,
Nantural non-toxic, active effect are good.
2. technical solution
To solve the above-mentioned problems, the technical solution adopted in the present invention is as follows:
A kind of dihydroxy isoquinolin, molecular formula C9H7NO2, it is 3,8- dihydroxy isoquinolin, molecular weight 161.1, knot
Structure formula are as follows:
A method of it extracting active component from centipede, the steps include:
A. centipede alcohol extract is prepared with the alcoholic solution that volumetric concentration is 45-65%；
B. using the centipede alcohol extract in Gel-filtration purification procedures a, multiple component samples are collected, are examined
It surveys and determines the wherein best component sample of anti-tumor activity, be denoted as component sample A, contain above-mentioned dihydroxy in component sample A
Isoquinolin.
Preferably, the step of centipede alcohol extract is prepared in step a are as follows:
(1) Centipede is prepared, the PBS solution that 5-10 times of Centipede volume is then added carries out ultrasonication homogenate；
(2) to homogenate refrigerated centrifuge, supernatant is taken, the centipede water extract being lyophilized；
(3) alcoholic solution for taking the obtained water extract in step (2) that 5-10 times of Centipede volume is added is homogenized, so
It is leached under the conditions of 4 DEG C afterwards, the volumetric concentration of alcoholic solution is 45-65%；
(4) to leaching liquid refrigerated centrifuge again, supernatant is taken, the centipede alcohol extract being lyophilized.
Preferably, the step of centipede alcohol extract being isolated and purified using Gel-filtration in step b are as follows:
(1) sephadex Sephadex G25 is injected in chromatographic column, is rinsed with mobile phase, mobile phase is 10~50%
Ethyl alcohol；
(2) centipede alcohol extract is configured to 20~50mg/mL, 4~8mL of loading in chromatographic column, flow velocity is 0.6~2mL/
Min collects each component sample according to chromatography map, carries out Activity determination, the best component of anti-tumor activity to each component sample
It is denoted as component sample A.
A method of above-mentioned dihydroxy isoquinolin is prepared, the steps include:
(a) centipede alcohol extract is prepared with the alcoholic solution that volumetric concentration is 45-65%；
(b) using the centipede alcohol extract in Gel-filtration purification procedures (a), multiple component samples are collected
Product, detect the anti-tumor activity of these component samples, and the best component of anti-tumor activity is denoted as component sample A；
(c) using component sample A obtained in preparative RP-HPLC purification procedures (b), gradient elution collection is carried out
Multiple Peak Activity components, detect the anti-tumor activity of these Peak Activity components, and the best component of anti-tumor activity is denoted as component B；
(d) it using the component B in semi-preparative RP-HPLC purification procedures (c), carries out gradient elution and collects multiple work
Property peak component, detects the anti-tumor activity of these Peak Activity components, the best component of anti-tumor activity is above-mentioned dihydroxy
Isoquinolin.
3,8- dihydroxy isoquinolin is preparing the application in anti-malaria medicaments, antibacterials and anti-tumor drug.
Preferably, the malaria is originating from the Plasmodium vivax, malariae, plasmodium falciparum for parasitizing human body
And Plasmodium ovale.
Preferably, the bacterium bag includes Escherichia coli, staphylococcus aureus, pseudomonas aeruginosa, first/hemolytic
Streptococcus, streptococcus pneumonia and bloodthirsty Bacillus influenzae.
Preferably, the tumour be incidence originating from people, brain, thyroid gland, pancreas, lungs, liver, oesophagus, stomach,
Mammary gland, kidney, gall-bladder, colon or rectum, ovary, uterus, cervix, prostate, bladder, testis primary or secondary cancer with
And sarcoma.
3. beneficial effect
Compared with the prior art, the invention has the benefit that
(1) present invention gropes by a large amount of experiment, has found noval chemical compound 3,8- dihydroxy isoquinoline from centipede for the first time
Quinoline and its application in anti-malarial, antibacterial and anti-tumor drug, the experimental results showed that, 3,8- dihydroxy isoquinolin can be used for
Malaria is treated, also has the function of sterilization, while entity tumor can also be treated；The malaria is to parasitize the tertian fever of human body
Protozoon, malariae, plasmodium falciparum and Plasmodium ovale；The bacterium is Escherichia coli, staphylococcus aureus, verdigris
Pseudomonad, first/beta hemolytic streptococcus, streptococcus pneumonia and bloodthirsty Bacillus influenzae；The tumour is originating from people's
Incidence, brain, thyroid gland, pancreas, lungs, liver, oesophagus, stomach, mammary gland, kidney, gall-bladder, colon or rectum, ovary, uterus,
The primary or secondary cancer and sarcoma of cervix, prostate, bladder, testis；
(2) present invention extracts Centipede by different methods, is mentioned using mtt assay to a variety of of Centipede
It takes liquid to carry out Activity determination, has found the optimal extract of anti-tumor activity, then use sephadex chromatography and preparative
The multiple means such as efficient liquid phase isolate and purify the extract, pass through efficient liquid phase, mass spectrum, hydrogen spectrum, carbon spectrum and the means such as infrared
Isolated compound is analyzed, has determined play anti-malarial, antibacterial and antitumor in centipede alcohol extract active component for the first time
Active main component is 3,8- dihydroxy isoquinolin；
(3) present invention is tested by many experiments, and it is best antitumor to show that ethyl alcohol leaching can be extracted from Centipede
Active component, remove or reduce has stronger anti-tumor activity, has good with antitumor unrelated component, the component after extraction
Good development prospect；
(4) 3,8- dihydroxy isoquinolin of the present invention is extracted from centipede obtains, and the centipede place of production is widely distributed, is easy to
Cultivation, can be used for extensive extraction separation and purification；
(5) 3,8- dihydroxy isoquinoline structure of the present invention is simple, is readily synthesized, and can be produced into large-scale production
This is cheap；
(6) present invention extracts from Centipede obtained 3,8- dihydroxy isoquinolin for the first time, with known iloquinoline derivative
It is different on substituent group or the position of substitution to close object, and inventor is by experimental results demonstrate this substances to have well
Anti-malarial, antibacterial and anti-tumor activity can be used for preparing anti-malarial, antibacterial and anti-tumor drug.
Detailed description of the invention
Attached drawing 1 is the HPLC map of active material I of the present invention, and main peak appearance time is 10.870min in figure, by analysis
It can determine that the purity of active material I reaches 95% or more, can be used for subsequent structural identification；
Attached drawing 2 is the high resolution mass spectrum figure (HRESIMS) of active material I of the present invention, from high resolution mass spectrum: molecular formula
C9H7NO2(m/z 162.0557[M+H]+, calcd for 162.0550), compound molecular weight 161.0557；
Attached drawing 3 be active material I of the present invention hydrogen spectrogram (1H-NMR), it can be seen that hydrogen in active material I from hydrogen spectrum
The number ratio of the type of atom chemistry environment and different chemical environment hydrogen atoms；
Attached drawing 4 is the carbon spectrogram of active material I of the present invention, can be seen that the number of carbon atom in active material I from carbon spectrum
The type of mesh and carbon atom；
Attached drawing 5 is the two-dimentional HMBC map of active material I of the present invention, be can be seen that in active material I from HMBC map
The hydrocarbon relationship of long-range coupling；
Attached drawing 6 is the two-dimentional HSQC map of active material I of the present invention, be can be seen that in active material I from HSQC map
The hydrocarbon relationship being connected directly；
Attached drawing 7 is the infared spectrum of active material I of the present invention, from the spy that can be seen that in active material I in infared spectrum
Levy peak；
Specific structure parsing is as follows:
Infared spectrum (attached drawing 7) provides data IR (KBr) vmax:3544cm-1, 2656cm-1, 1687cm-1, 1598cm-1,
1575cm-1, 1361cm-1, 1248cm-1, 1201cm-1, 1131cm-1, 848cm-1, 795cm-1, 724cm-1, can go out to determine the work
Property substance I there is benzene ring structure, have hydroxyl in substituent group.High resolution mass spectrum HRESIMS (attached drawing 2) provides molecular formula C9H7NO2
(m/z 162.0557[M+H]+, calcd for 162.0550), illustrate that active material I has 7 degrees of unsaturation.Attached drawing 31H-NMR(D2O, 300MHz) in δH7.29 (1H, d, J=7.8Hz, H-5), 7.50 (1H, t, J=7.2Hz, H-6), 7.07 (1H,
D, J=7.1Hz, H-7), it is 3 proton signals of ABX system on phenyl ring, δH8.42 (1H, s, H-1), 8.02 (1H, s, H-4)
In all to be unimodal.Attached drawing 413C-NMR(D2O, 75MHz), δC: 153.5 (C-3), 149.3 (C-8), signal show the compound
Middle C-3 and C-8 are connected with hydroxyl.Binding molecule formula determines that active material I is dihydroxy quinoline or dihydroxy iloquinoline derivative chemical combination
Object, one of hydroxyl is located at pyridine ring 3, and perhaps 4 another hydroxyls are located at phenyl ring 5 or 8.At HSQC (attached drawing 6)
In it can be seen that δC: 136.9 (C-1), 133.0 (C-6), 129.9 (C-4), 120.2 (C-5), 115.5 (C-7) are directly former with hydrogen
Son is connected.In HMBC (attached drawing 5), δH8.42 (1H, s, H-1) and δC129.9 (C-4) and δCThere are long-range phases by 126.8 (C-9)
It closes；δH8.02 (1H, s, H-4) and δC136.9 (C-1), δC153.5 (C-3), δC120.2 (C-5) and δC126.8 (C-9) exist far
Cheng Xiangguan；δH7.29 (1H, d, J=7.8Hz, H-5) and δC129.9 (C-4), δC133.0 (C-6), δC115.5 (C-7) and δCThere are long-range correlations by 149.3 (C-8), this illustrates that the compound is isoquinoline compound, and 2 hydroxyls are respectively 3 and 8
Replace.The data analysis result of comprehensive attached drawing 1-7 can determine that active material I is 3,8- dihydroxy isoquinolin, and molecular weight is
161.1 structural formula are as follows:
Attached drawing 8 is the sephadex chromatography map of centipede alcohol extract, has collected 3 components (component 1~3) altogether；
Attached drawing 9 is the preparative efficient liquid phase separating spectrum of sephadex chromatography component 3, has collected 6 component (groups altogether
Divide 3-1~3-6)；
Attached drawing 10 is the Semi-preparative High Performance liquid phase separation map of component 3-2, and 2 samples (sample 1~2) is obtained,
Middle sample 2 is 3,8- dihydroxy isoquinolin.
Specific embodiment
Embodiment 1
The preparation of 3,8- dihydroxy isoquinolin and Structural Identification in centipede
A. water extraction and alcohol extracting method prepare Scolopendra extract:
(1) centipede stem body is crushed to obtain Centipede, 10 times of Centipede volume (5-10 times of volume, sheet is added
Preferably 10 times in embodiment, behind similarly) PBS homogenate, then carry out ultrasonication；
(2) refrigerated centrifuge is carried out to homogenate, takes supernatant, freeze-drying obtains centipede water extract
(3) it takes and is precipitated obtained in step (2), 10 times of precipitation volume of 55% (volume fraction, subsequent similarly) is added
Ethyl alcohol homogenate, then leaches under the conditions of 4 DEG C, to leaching liquid refrigerated centrifuge again, takes supernatant, freeze-drying obtains centipede
55% alcohol extract；
(4) it takes and is precipitated obtained in step (2), 10 times of precipitation volume of 70% ethyl alcohol homogenate is added, then in 4 DEG C of conditions
Lower leaching takes supernatant to leaching liquid refrigerated centrifuge again, and freeze-drying obtains 70% alcohol extract of centipede；
(5) it takes and is precipitated obtained in step (2), 10 times of precipitation volume of 85% ethyl alcohol homogenate is added, then in 4 DEG C of conditions
Lower leaching takes supernatant to leaching liquid refrigerated centrifuge again, and freeze-drying obtains 85% alcohol extract of centipede；
(6) MTT detects the anti tumor activity in vitro of centipede water extract and alcohol extract, the results are shown in Table 1；
1 centipede water extract of table and alcohol extract act on 5 plants of Cytostatic to tumor cell
According to MTT Activity determination as a result, the anti tumor activity in vitro of discovery 55% alcohol extract of centipede is best, and it presses down
Make the IC of 5 plants of tumor cell proliferations50Value difference is away from smaller.Relative to 55% alcohol extract of centipede, water extract, 70% alcohol extract and 85%
The anti tumor activity in vitro of alcohol extract is poor.55% alcohol extract of centipede is isolated and purified in subsequent implementation method.
B. active component in Gel-filtration preparation 55% alcohol extract of centipede
(1) chromatographic column (specification: internal diameter 10mm* height 500mm) is mounted on iron stand, and by itself and digital display constant flow pump
And the connection of nucleic acid-protein detector, chromatographic flow mutually select 10% ethyl alcohol；
(2) sephadex Sephadex G25 heats 4h in boiling water bath, and taking-up is cooled to room temperature, and then will
Sephadex G25 is added continuously in chromatographic column, and is balanced with the mobile phase of 4 bed volumes；
(3) 55% alcohol extract of centipede is configured to 50mg/mL, the loading 4mL in chromatographic column, flow velocity 2mL/min.It opens
Nucleic acid-protein detector test, and each component sample is collected according to chromatography map, 3 components (component 1~3) are collected altogether, specifically
See attached drawing 8.Mtt assay detection is carried out to 3 components, the results are shown in Table 2；
2 component of table, 1~3 pair of 5 plants of Cytostatic to tumor cell effect
According to MTT Activity determination as a result, the anti tumor activity in vitro of discovery component 3 is best, and it inhibits 5 plants of tumours
The IC of cell Proliferation50Value difference is away from smaller.Relative to component 3, the anti tumor activity in vitro of component 1 and component 2 is obviously poor.It is subsequent
Implementation method in component 3 is isolated and purified.
C. high performance liquid chromatography prepares 3,8- dihydroxy isoquinolin
(1) sephadex chromatography component 3 is isolated and purified with preparative RP-HPLC.It is flat with 8% acetonitrile (containing 0.1%TFA)
It weighs after 1h, carries out gradient elution, elute final concentration of 35%, elution time 50min, flow velocity 50mL/min, collect activity
Peak, freeze-drying collect 6 components (component 3-1~3-6) altogether, are specifically shown in attached drawing 9；Mtt assay detection, knot are carried out to 6 components
Fruit is shown in Table 3；
3 component 3-1~3-6 of table acts on 3 plants of Cytostatic to tumor cell
According to MTT Activity determination as a result, the anti tumor activity in vitro of discovery component 3-2 is best, and it inhibits 3 plants to swell
The IC of tumor cell proliferation50Value difference is away from smaller.Component 3-2 is isolated and purified in subsequent implementation method.
(2) secondarily purified to component 3-2 progress in step (1), it is isolated and purified with semi-preparative RP-HPLC.With 8% acetonitrile
After (contain 0.1%TFA) balance 1h, gradient elution is carried out, elution final concentration of 30%, elution time 30min, flow velocity is
10mL/min collects Peak Activity, and freeze-drying, purifying obtains 2 samples (sample 1~2) from component 3-2, as shown in Figure 10；
Mtt assay detection is carried out to 2 samples, the results are shown in Table 4.
4 sample of table, 1~2 pair of 3 plants of Cytostatic to tumor cell effect
According to MTT Activity determination as a result, the anti tumor activity in vitro of discovery sample 2 is best, and it inhibits 3 plants of tumours
The IC of cell Proliferation50Value difference is away from smaller, and the anti tumor activity in vitro of sample 1 is poor, therefore sample 2 (attached drawing 10) in component 3-2
It is denoted as active material I, purity detecting, Structural Identification and work are carried out to active material I in subsequent implementation method and embodiment
Property investigate.
(3) content and purity of active material I are analyzed by analytic type RP-HPLC.It is flat with 8% acetonitrile (containing 0.5%TFA)
It weighs after 1h, carries out gradient elution, elute final concentration of 30%, elution time 30min, flow velocity 1mL/min, as shown in Fig. 1,
The appearance time of active material I is 10.870min；
(4) infrared spectroscopy, mass spectrum, carbon spectrum, hydrogen spectrum, two-dimentional spectrum analysis are carried out to active material I, determines its structure, it is such as attached
Shown in Fig. 2-7, active material I is 3,8- dihydroxy isoquinolin (isoquinoline-3,8-diol), molecular formula C9H7NO2,
Molecular weight is 161.1, structural formula are as follows:
Embodiment 2
Substantially with embodiment 1 the step of extraction active component in the present embodiment, except that: 1) when water mentions, addition
PBS solution volume is 5 times of Centipede volume, and when alcohol extracting, the volume fraction of alcoholic solution is 45%；2) sephadex is used
When chromatography isolates and purifies centipede alcohol extract, centipede alcohol extract is configured to 20mg/mL, mobile phase is 50% ethyl alcohol, loading body
Product is 8mL, flow velocity 0.6mL/min.
The result of the present embodiment is 3,8- dihydroxy isoquinoline through Structural Identification with embodiment 1, finally obtained active material
Quinoline (isoquinoline-3,8-diol), molecular formula C9H7NO2, molecular weight 161.1, structural formula are as follows:
Embodiment 3
Substantially with embodiment 1 the step of extraction active component in the present embodiment, except that: 1) when water mentions, addition
PBS solution volume is 8 times of Centipede volume, and when alcohol extracting, the volume fraction of alcoholic solution is 65%；2) sephadex is used
When chromatography isolates and purifies centipede alcohol extract, centipede alcohol extract is configured to 35mg/mL, mobile phase is 30% ethyl alcohol, loading body
Product is 6mL, flow velocity 1.5mL/min.
The result of the present embodiment is 3,8- dihydroxy isoquinoline through Structural Identification with embodiment 1, finally obtained active material
Quinoline (isoquinoline-3,8-diol), molecular formula C9H7NO2, molecular weight 161.1, structural formula are as follows:
Embodiment 4
SYBR Green I method evaluates the external anti-malarial activity of 3,8- dihydroxy isoquinolin
(1) plasmodium: Plasmodium vivax, malariae, plasmodium falciparum and Plasmodium ovale；
(2) positive drug: chloroquine；
(3) test method: the lysate prepared by fluorescent dye SYBR Green I and in advance is with 0.20 μ L.mL-1It is dense
Degree ratio is uniformly mixed, and is protected from light.When polypide growth conditions reach ring bodies period infection rate > 70% (avoid trophozoite period and
Schizont period) when collect polypide, be 2% with complete medium tune hematocrit, protozoan infection rate requires as 0.3%~
0.5%.Add 20 μ L pastille culture medium, 80 μ L worm blood in the every hole of 96 orifice plates, if blank well (red blood cell of 2% hematocrit) and control wells
(2% hematocrit, the worm blood of 0.3%~0.5% infection rate).Continue to cultivate, freeze medicine plate in -20 DEG C of refrigerators after 72h, overnight.
Every kind of drug sets 8 concentration gradients, and each concentration sets 3 multiple holes, and experiment is repeated 3 times every time.Medicine plate is placed at room temperature for 2h by next day,
After Kong Zhonghan worm blood dissolves completely, the lysate of the I of Green containing SYBR is added in the ratio of 1:1.It is protected from light in darkroom and places 1h
Afterwards, 100 μ L of absorption are mixed and are transferred to 96 hole black microwell plates.Medicine plate is put into all-wave length multi-function microplate reader, in 485nm
(Ex) and under the conditions of 535nm (Em) fluorescent value in every hole in plate is read.Data (different pharmaceutical concentration is arranged using Excel software
And its fluorescent value) software processing, after the data obtained is normalized, the curve matching of nonlinear regression is carried out, obtains each antimalarial
Inhibiting rate and IC50Value, determines the antimalarial active of each drug.
(4) experimental data is analyzed using statistic software SPSS, measurement data data are with (mean ± standard deviation) table
Show, using variance analysis, compare examined using LSD-t two-by-two, is that difference is statistically significant with p < 0.05.
5 3,8- dihydroxy isoquinolin of table detects the anti-malarial external activity of 4 kinds of plasmodiums
As a result: being shown in Table 5,3,8- dihydroxy isoquinolin to Plasmodium vivax, malariae, plasmodium falciparum and ovale malaria
The breeding of protozoon has good inhibiting effect, IC50Value be respectively 26.27 μ g/mL, 17.74 μ g/mL, 37.11 μ g/mL and
18.80μg/mL.3,8- dihydroxy isoquinolin are preferable to the antimalarial active of malariae and Plasmodium ovale, to tertian fever original
The antimalarial active of worm and plasmodium falciparum is poor.Compared with positive drug chloroquine, 3,8- dihydroxy isoquinolin are to malariae
Antimalarial active is higher.Above the experimental results showed that, growth of 3, the 8- dihydroxy isoquinolin to malariae and Plasmodium ovale
With stronger inhibiting effect, can be used for preparing anti-malaria medicaments.
Embodiment 5
Microdilution plate method evaluates 3,8- dihydroxy isoquinolin antibacterial activity in vitro
(1) bacterial strain: Escherichia coli, staphylococcus aureus, pseudomonas aeruginosa, first/beta hemolytic streptococcus, pneumonia
Streptococcus and bloodthirsty Bacillus influenzae.
(2) positive drug: streptomycin sulphate.
(3) test method: it is 2 × 10 that concentration is added into 96 well culture plates of cleaning sterile6Cfu/mL for try bacterium solution
90 μ L, are then added the 10 μ L of test sample group mother liquor of various concentration gradient, and positive controls are the sulfuric acid strepto- of various concentration
10 μ L of element.Blank control and solvent control (30% ethyl alcohol or 30%DMSO or 30% acetone), each group of 4 multiple holes are set simultaneously.
Concentration of the compound in bacterium solution is 1/10th of mother liquid concentration.By 96 orifice plates at 37 DEG C, 5%CO2It is trained in incubator
It supports for 24 hours, observes by the naked eye, the lowest concentration of drug in the completely limpid hole of solution is MIC value.
6 3,8- dihydroxy isoquinolin of table detects the antibacterial external activity of 6 kinds of bacterial strains
As a result: be shown in Table 6,3,8- dihydroxy isoquinolin to the breeding of staphylococcus aureus and bloodthirsty Bacillus influenzae have compared with
Good inhibiting effect, MIC value is respectively 32 μ g/mL and 16 μ g/mL.3,8- dihydroxy isoquinolin to staphylococcus aureus and
The antibacterial activity of bloodthirsty Bacillus influenzae is preferable, to Escherichia coli, pseudomonas aeruginosa, first/beta hemolytic streptococcus and pneumonia
Streptococcic antibacterial activity is general.3,8- dihydroxy isoquinolin is to staphylococcus aureus, first/beta hemolytic streptococcus and lung
Scorching streptococcic antibacterial activity is higher than positive drug streptomycin sulphate, to the antibacterial activity of pseudomonas aeruginosa and bloodthirsty Bacillus influenzae
It is suitable with positive drug streptomycin sulphate.The experimental results showed that, 3,8- dihydroxy isoquinolin are to staphylococcus aureus and thermophilic above
The breeding of blood Bacillus influenzae has stronger inhibiting effect, and MIC value is respectively less than 64 μ g/mL, can be used for preparing antibacterials.
Embodiment 6
Mtt assay evaluates 3,8- dihydroxy isoquinolin anti tumor activity in vitro
(1) tumor cell line: human cervical carcinoma Hela, human liver cancer Bel-7402, human prostata cancer DU-145, people's kidney
A498, human large cell lung cancer H460, human nasopharyngeal carcinoma CNE, human esophagus cancer EC109, human thyroid squamous carcinoma SW-579, human pancreas cancer
SW-1990, human breast carcinoma MDA-MB-231, Human gallbladder carcinoma GBC-SD, human colon carcinoma HT-29, human ovarian cancer SK-OV-3, people's
Endometrial carcinoma HHUA, human bladder cancer HT1376, Human Testis cancer 5637, people's sarcoma HT-1080 and human gastric cancer MGC-803 cell；
(2) positive control drug: 10 μ g/mL of taxol；
(3) 3,8- dihydroxy isoquinolin: the solution that blank cultures are configured to 1mg/mL is placed and -80 DEG C of preservations.Face use
Before, final concentration of 1 μ g/mL, 2 μ g/mL, 4 μ g/mL, 8 μ g/mL, 16 μ g/mL, 32 μ g/mL, 64 μ are diluted to blank cultures
The solution of g/mL.
(4) experimental method: mtt assay
(5) human cervical carcinoma Hela, human liver cancer Bel-7402, human prostata cancer DU-145, people's kidney A498, National People's Congress's cell lung
Cancer H460, human nasopharyngeal carcinoma CNE, human esophagus cancer EC109, human thyroid squamous carcinoma SW-579, human pancreas cancer SW-1990, human breast carcinoma
MDA-MB-231, Human gallbladder carcinoma GBC-SD, human colon carcinoma HT-29, human ovarian cancer SK-OV-3, people's carcinoma of endometrium HHUA, people's wing
Guang cancer HT1376, Human Testis cancer 5637, people's sarcoma HT-1080 and human gastric cancer MGC-803 cell are trained with the DMEM containing 10%FBS
Base is supported, in 37 DEG C, 5%CO2Incubator in culture to 90% or more convergence degree when, with trypsin digestion, 800rpm ×
5min is collected by centrifugation, and cell is resuspended with the DMEM containing 10%FBS, and counting under the microscope and adjusting cell concentration is 3 × 104
A/mL, cell inoculation is into 96 orifice plates, every 100 μ L of hole.Cell into 96 orifice plates is inoculated at 37 DEG C, 5%CO2It is trained in incubator
It supports overnight, it is completely adherent to cell.As dosing group, taxol is not added and appoints as positive controls 3,8- dihydroxy isoquinolin
The blank cultures of what drug are added in 96 orifice plates as negative control group.By 96 orifice plates at 37 DEG C, 5%CO2It is incubated in incubator
Educate 48h.MTT, the every 20 μ L of hole of 5mg/mL are added into 96 orifice plates, continues to cultivate 4h in incubator.It discards in 96 orifice plates
Culture solution, every hole are added 100 μ L DMSO, mix gently.It is surveyed at measurement wavelength 570nm, reference wavelength 630nm with microplate reader
Determine light absorption value.Proliferation inhibition rate (Proliferation inhibition rate, PI) is calculated according to formula:
(7) concentration for utilizing proliferation inhibition rate and dosing group, calculates half-inhibitory concentration IC50 using SPSS software, such as
Shown in table 1.Test obtained result is indicated with mean ± SD, and is carried out statistics T and examined, and * P < 0.05 is significant difference, * * P
< 0.01 is extremely significant sex differernce.
7 3,8- dihydroxy isoquinolin of table detects the antitumor external activity of 18 kinds of tumor cell lines
As a result: being shown in Table 7,3,8- dihydroxy isoquinolin to people's kidney A498, human large cell lung cancer H460, human breast carcinoma MDA-
MB-231, human colon carcinoma HT-29, human bladder cancer HT1376 and people's sarcoma HT-1080 cell proliferation have and preferable inhibit to make
With IC50Value is respectively less than 10 μ g/mL, and the above experimental result illustrates 3,8- dihydroxy isoquinolin to the proliferation of these tumour cells
With stronger inhibiting effect, can be used for preparing anti-tumor drug.
Embodiment 7
3,8- dihydroxy isoquinolin is to human gastric cancer MGC-803 nude mouse xenograft tumor growth inhibition effect
By 3,8- dihydroxy isoquinolin according to 100mg/kg/1d (height), 70mg/kg/1d (in), 40mg/kg/1d (low) gives
Pharmaceutical quantities, in mouse bare subcutaneous injection, once a day, 0.2mL/ times；Using taxol as positive controls 10mg/kg, in nude mice skin
Lower injection, once a week.
The tumor tissue of growth animated period is taken to cut into 1.5mm3Left and right is aseptically inoculated in skin on the right side of nude mouse
Under.Mice-transplanted tumor vernier caliper measurement transplantable tumor diameter, to tumour growth to 60-80mm3Animal is grouped at random afterwards.It uses
The method for measuring knurl footpath, dynamic observation are tested the effect of drugs against tumor.The pendulous frequency of diameter of tumor is every 2 days 1 time, every time
Measurement also needs to weigh mouse weight simultaneously.Experimental group injects 3,8- dihydroxy isoquinolin, and feminine gender is organized while to same amount of normal saline.
Gross tumor volume calculation formula: TV=0.52 × a × b2；
Wherein, a, b respectively indicate length and width.Relative tumour volume (relative tumor is calculated according to the result of measurement
Volume, RTV), calculation formula are as follows: RTV=Vt/V0, wherein V0 is that resulting gross tumor volume is measured when dividing cage, and Vt is to test
The gross tumor volume measured each time in journey.The evaluation index of anti-tumor activity is Relative tumor proliferation rate T/C (%), and formula is such as
Under:
T/C (%)=TRTV/CRTV× 100%
TRTV: treatment group RTV；CRTV: negative control group RTV.
The inhibiting effect that 8 3,8- dihydroxy isoquinolin of table grows MGC-803 nude mouse xenograft tumor
As a result: being shown in Table 8, taxol 10mg/kg, the tumour inhibiting rate to MGC-803 Nude Mice is 54.90%, to reality
The weight for testing animal has the influence of conspicuousness；High, normal, basic three groups of 3,8- dihydroxy isoquinolin to MGC-803 Nude Mice
Tumour inhibiting rate be 46.58%, 34.67%, 30.61%.
3,8- dihydroxy isoquinolin to MGC-803 Nude Mice growth inhibition test the result shows that, with negative control
Group is compared, and 3 groups of dosing groups have the inhibiting effect of conspicuousness to the growth of MGC-803 transplantable tumor, and each experimental mice weight is omited
There is reduction；Each dosing group and positive drug group have dead mouse.
Embodiment 8
3,8- dihydroxy isoquinolin is to human breast carcinoma MDA-MB-231 nude mouse xenograft tumor growth inhibition effect
By 3,8- dihydroxy isoquinolin according to 100mg/kg/1d (height), 70mg/kg/1d (in), 40mg/kg/1d (low) gives
Pharmaceutical quantities, in mouse bare subcutaneous injection, once a day, 0.2mL/ times；Taxol is set as positive controls 10mg/kg, in nude mice
Subcutaneous injection, once a week.
The tumor tissue of growth animated period is taken to cut into 1.5mm3Left and right is aseptically inoculated in skin on the right side of nude mouse
Under.Mice-transplanted tumor vernier caliper measurement transplantable tumor diameter, to tumour growth to 60-80mm3Animal is grouped at random afterwards.It uses
The method for measuring knurl footpath, dynamic observation are tested the effect of drugs against tumor.The pendulous frequency of diameter of tumor is every 2 days 1 time, every time
Measurement also needs to weigh mouse weight simultaneously.Experimental group injects 3,8- dihydroxy isoquinolin, and feminine gender is organized while to same amount of normal saline.
Gross tumor volume calculation formula: TV=0.52 × a × b2；
Wherein, a, b respectively indicate length and width.Relative tumour volume (relative tumor is calculated according to the result of measurement
Volume, RTV), calculation formula are as follows: RTV=Vt/V0, wherein V0 is that resulting gross tumor volume is measured when dividing cage, and Vt is to test
The gross tumor volume measured each time in journey.The evaluation index of anti-tumor activity is Relative tumor proliferation rate T/C (%), and formula is such as
Under:
T/C (%)=TRTV/CRTV× 100%
TRTV: treatment group RTV；CRTV: negative control group RTV.
The inhibiting effect that 9 3,8- dihydroxy isoquinolin of table grows MDA-MB-231 nude mouse xenograft tumor
As a result: being shown in Table 9, taxol 10mg/kg, the tumour inhibiting rate to MDA-MB-231 Nude Mice is 77.95%, right
The weight of experimental animal has the influence of conspicuousness；High, medium and low three groups of 3,8- dihydroxy isoquinolin to MDA-MB-231 nude mouse
The tumour inhibiting rate of transplantable tumor is 64.65%, 55.65%, 48.22%.
3,8- dihydroxy isoquinolin to MDA-MB-231 Nude Mice growth inhibition test the result shows that, it is right with feminine gender
It is compared according to group, 3 groups of dosing groups have the inhibiting effect of conspicuousness to the growth of MDA-MB-231 transplantable tumor.Each experimental mice weight
It slightly reduces, each dosing group and positive drug group have dead mouse.
Embodiment 9
3,8- dihydroxy isoquinolin is to human liver cancer Bel-7402 nude mouse xenograft tumor growth inhibition effect
By 3,8- dihydroxy isoquinolin according to 100mg/kg/1d (height), 70mg/kg/1d (in), 40mg/kg/1d (low) gives
Pharmaceutical quantities, in mouse bare subcutaneous injection, once a day, 0.2mL/ times；Taxol is set as positive controls 10mg/kg, in nude mice
Subcutaneous injection, once a week.
The tumor tissue of growth animated period is taken to cut into 1.5mm3Left and right is aseptically inoculated in skin on the right side of nude mouse
Under.Mice-transplanted tumor vernier caliper measurement transplantable tumor diameter, to tumour growth to 60-80mm3Animal is grouped at random afterwards.It uses
The method for measuring knurl footpath, dynamic observation are tested the effect of drugs against tumor.The pendulous frequency of diameter of tumor is every 2 days 1 time, every time
Measurement also needs to weigh mouse weight simultaneously.Experimental group injects 3,8- dihydroxy isoquinolin, and feminine gender is organized while to same amount of normal saline.
Gross tumor volume calculation formula: TV=0.52 × a × b2；
Wherein, a, b respectively indicate length and width.Relative tumour volume (relative tumor is calculated according to the result of measurement
Volume, RTV), calculation formula are as follows: RTV=Vt/V0, wherein V0 is that resulting gross tumor volume is measured when dividing cage, and Vt is each time
The gross tumor volume of measurement.The evaluation index of anti-tumor activity is Relative tumor proliferation rate T/C (%), and formula is as follows:
T/C (%)=TRTV/CRTV× 100%
TRTV: treatment group RTV；CRTV: negative control group RTV.
The inhibiting effect that 10 3,8- dihydroxy isoquinolin of table grows Bel-7402 nude mouse xenograft tumor
As a result: being shown in Table 10, taxol 10mg/kg, the tumour inhibiting rate to Bel-7402 Nude Mice is 74.73%, right
The weight of experimental animal has the influence of conspicuousness；High, medium and low three groups of 3,8- dihydroxy isoquinolin are moved Bel-7402 nude mouse
The tumour inhibiting rate for planting tumor is 58.28%, 49.35%, 30.73%.
3,8- dihydroxy isoquinolin to Bel-7402 Nude Mice growth inhibition test the result shows that, with negative control
Group is compared, and 3 groups of dosing groups have the inhibiting effect of conspicuousness to the growth of Bel-7402 transplantable tumor.Each experimental mice weight is omited
Have a reduction, in 3,8- dihydroxy isoquinolin, low group have mouse dead without dead mouse phenomenon, other dosing groups and positive drug group
It dies.
Embodiment 10
3,8- dihydroxy isoquinolin is to human lung cancer H-460 nude mouse xenograft tumor growth inhibition effect
By 3,8- dihydroxy isoquinolin according to 100mg/kg/1d (height), 70mg/kg/1d (in), 40mg/kg/1d (low) gives
Pharmaceutical quantities, in mouse bare subcutaneous injection, once a day, 0.2mL/ times；Taxol is set as positive controls 10mg/kg, in nude mice
Subcutaneous injection, once a week.
The tumor tissue of growth animated period is taken to cut into 1.5mm3Left and right is aseptically inoculated in skin on the right side of nude mouse
Under.Mice-transplanted tumor vernier caliper measurement transplantable tumor diameter, to tumour growth to 60-80mm3Animal is grouped at random afterwards.It uses
The method for measuring knurl footpath, dynamic observation are tested the effect of drugs against tumor.The pendulous frequency of diameter of tumor is every 2 days 1 time, every time
Measurement also needs to weigh mouse weight simultaneously.Experimental group injects 3,8- dihydroxy isoquinolin, and feminine gender is organized while to same amount of normal saline.
Gross tumor volume calculation formula: TV=0.52 × a × b2；
Wherein, a, b respectively indicate length and width.Relative tumour volume (relative tumor is calculated according to the result of measurement
Volume, RTV), calculation formula are as follows: RTV=Vt/V0, wherein V0 is that resulting gross tumor volume is measured when dividing cage, and Vt is each time
The gross tumor volume of measurement.The evaluation index of anti-tumor activity is Relative tumor proliferation rate T/C (%), and formula is as follows:
T/C (%)=TRTV/CRTV× 100%
TRTV: treatment group RTV；CRTV: negative control group RTV.
The inhibiting effect that 11 3,8- dihydroxy isoquinolin of table grows H-460 nude mouse xenograft tumor
As a result: being shown in Table 11, taxol 10mg/kg, the tumour inhibiting rate to H-460 Nude Mice is 68.35%, to experiment
The weight of animal has the influence of conspicuousness；High, medium and low three groups of 3,8- dihydroxy isoquinolin to H-460 Nude Mice
Tumour inhibiting rate is 59.81%, 50.74%, 45.03%.
3,8- dihydroxy isoquinolin to H-460 Nude Mice growth inhibition test the result shows that, with negative control group
It compares, 3 groups of dosing groups have the inhibiting effect of conspicuousness to the growth of H-460 transplantable tumor.Each experimental mice weight slightly drops
Low, 3,8- low group of dihydroxy isoquinolin have dead mouse without dead mouse phenomenon, other dosing groups and positive drug group.
Embodiment 11
3,8- dihydroxy isoquinolin is to human thyroid carcinomas SW-579 nude mouse xenograft tumor growth inhibition effect
By 3,8- dihydroxy isoquinolin according to 100mg/kg/1d (height), 70mg/kg/1d (in), 40mg/kg/1d (low) gives
Pharmaceutical quantities, in mouse bare subcutaneous injection, once a day, 0.2mL/ times；Taxol is set as positive controls 10mg/kg, in nude mice
Subcutaneous injection, once a week.
The tumor tissue of growth animated period is taken to cut into 1.5mm3Left and right is aseptically inoculated in skin on the right side of nude mouse
Under.Mice-transplanted tumor vernier caliper measurement transplantable tumor diameter, to tumour growth to 60-80mm3Animal is grouped at random afterwards.It uses
The method for measuring knurl footpath, dynamic observation are tested the effect of drugs against tumor.The pendulous frequency of diameter of tumor is every 2 days 1 time, every time
Measurement also needs to weigh mouse weight simultaneously.Experimental group injects 3,8- dihydroxy isoquinolin, and feminine gender is organized while to same amount of normal saline.
Gross tumor volume calculation formula: TV=0.52 × a × b2；
Wherein, a, b respectively indicate length and width.Relative tumour volume (relative tumor is calculated according to the result of measurement
Volume, RTV), calculation formula are as follows: RTV=Vt/V0, wherein V0 is that resulting gross tumor volume is measured when dividing cage, and Vt is each time
The gross tumor volume of measurement.The evaluation index of anti-tumor activity is Relative tumor proliferation rate T/C (%), and formula is as follows:
T/C (%)=TRTV/CRTV× 100%
TRTV: treatment group RTV；CRTV: negative control group RTV.
The inhibiting effect that 12 3,8- dihydroxy isoquinolin of table grows SW-579 nude mouse xenograft tumor
As a result: being shown in Table 12, taxol 10mg/kg, the tumour inhibiting rate to SW-579 Nude Mice is 66.64%, to reality
The weight for testing animal has the influence of conspicuousness；High, medium and low three groups of 3,8- dihydroxy isoquinolin to SW-579 Nude Mice
Tumour inhibiting rate be 60.28%, 50.41%, 45.62%%.
3,8- dihydroxy isoquinolin to SW-579 Nude Mice growth inhibition test the result shows that, with negative control group
It compares, high, medium and low three groups of 3, the 8- dihydroxy isoquinolin inhibiting effect for having conspicuousness to the growth of SW-579 transplantable tumor.Each reality
Testing group mouse weight slightly reduces, and dosing group and positive drug group are without dead mouse phenomenon.
Embodiment 12
3,8- dihydroxy isoquinolin is to human colon carcinoma HT-29 nude mouse xenograft tumor growth inhibition effect
By 3,8- dihydroxy isoquinolin according to 100mg/kg/1d (height), 70mg/kg/1d (in), 40mg/kg/1d (low) gives
Pharmaceutical quantities, in mouse bare subcutaneous injection, once a day, 0.2mL/ times；Taxol is set as positive controls 10mg/kg, in nude mice
Subcutaneous injection, once a week.
The tumor tissue of growth animated period is taken to cut into 1.5mm3Left and right is aseptically inoculated in skin on the right side of nude mouse
Under.Mice-transplanted tumor vernier caliper measurement transplantable tumor diameter, to tumour growth to 60-80mm3Animal is grouped at random afterwards.It uses
The method for measuring knurl footpath, dynamic observation are tested the effect of drugs against tumor.The pendulous frequency of diameter of tumor is every 2 days 1 time, every time
Measurement also needs to weigh mouse weight simultaneously.Experimental group injects 3,8- dihydroxy isoquinolin, and feminine gender is organized while to same amount of normal saline.
Gross tumor volume calculation formula: TV=0.52 × a × b2；
Wherein, a, b respectively indicate length and width.Relative tumour volume (relative tumor is calculated according to the result of measurement
Volume, RTV), calculation formula are as follows: RTV=Vt/V0, wherein V0 is that resulting gross tumor volume is measured when dividing cage, and Vt is each time
The gross tumor volume of measurement.The evaluation index of anti-tumor activity is Relative tumor proliferation rate T/C (%), and formula is as follows:
T/C (%)=TRTV/CRTV× 100%
TRTV: treatment group RTV；CRTV: negative control group RTV.
The inhibiting effect that 13 3,8- dihydroxy isoquinolin of table grows HT-29 nude mouse xenograft tumor
As a result: being shown in Table 13, taxol 10mg/kg, the tumour inhibiting rate to HT-29 Nude Mice is 68.74%, to experiment
The weight of animal has the influence of conspicuousness；High, medium and low three groups of 3,8- dihydroxy isoquinolin to HT-29 Nude Mice
Tumour inhibiting rate is 59.78%, 52.95%, 47.29%.
3,8- dihydroxy isoquinolin to HT-29 Nude Mice growth inhibition test the result shows that, with negative control group
It compares, 3 groups of dosing groups have the inhibiting effect of conspicuousness to the growth of HT-29 transplantable tumor.Each experimental mice weight slightly drops
It is low, in 3,8- dihydroxy isoquinolin, low group of mouse without the phenomena of mortality, other dosing groups and positive drug group mouse have death.
Embodiment 13
3,8- dihydroxy isoquinolin is to human cervical carcinoma's Hela nude mouse xenograft tumor growth inhibition effect
By 3,8- dihydroxy isoquinolin according to 100mg/kg/1d (height), 70mg/kg/1d (in), 40mg/kg/1d (low) gives
Pharmaceutical quantities, in mouse bare subcutaneous injection, once a day, 0.2mL/ times；Taxol is set as positive controls 10mg/kg, in nude mice
Subcutaneous injection, once a week.
The tumor tissue of growth animated period is taken to cut into 1.5mm3Left and right is aseptically inoculated in skin on the right side of nude mouse
Under.Mice-transplanted tumor vernier caliper measurement transplantable tumor diameter, to tumour growth to 60-80mm3Animal is grouped at random afterwards.It uses
The method for measuring knurl footpath, dynamic observation are tested the effect of drugs against tumor.The pendulous frequency of diameter of tumor is every 2 days 1 time, every time
Measurement also needs to weigh mouse weight simultaneously.Experimental group injects 3,8- dihydroxy isoquinolin, and feminine gender is organized while to same amount of normal saline.
Gross tumor volume calculation formula: TV=0.52 × a × b2；
Wherein, a, b respectively indicate length and width.Relative tumour volume (relative tumor is calculated according to the result of measurement
Volume, RTV), calculation formula are as follows: RTV=Vt/V0, wherein V0 is that resulting gross tumor volume is measured when dividing cage, and Vt is each time
The gross tumor volume of measurement.The evaluation index of anti-tumor activity is Relative tumor proliferation rate T/C (%), and formula is as follows:
T/C (%)=TRTV/CRTV× 100%
TRTV: treatment group RTV；CRTV: negative control group RTV.
The inhibiting effect that 14 3,8- dihydroxy isoquinolin of table grows Hela nude mouse xenograft tumor
As a result: being shown in Table 14, taxol 10mg/kg, the tumour inhibiting rate to Hela Nude Mice is 72.12%, to experiment
The weight of animal has the influence of conspicuousness；The high, medium and low three groups of suppressions to Hela Nude Mice of 3,8- dihydroxy isoquinolin
Ratio of outflow is 62.32%, 50.22%, 43.95%.
3,8- dihydroxy isoquinolin to Hela Nude Mice growth inhibition test the result shows that, with negative control group phase
Than high, normal, basic three groups of 3, the 8- dihydroxy isoquinolin inhibiting effect for having conspicuousness to the growth of Hela transplantable tumor.Each experimental group is small
Mouse weight slightly reduces, and dosing group has dead mouse without dead mouse phenomenon, positive drug group.
By embodiment 4~11, those skilled in the art can be found that 3,8- dihydroxy isoquinolin is preparing antineoplastic
Application in object, incidence, brain, thyroid gland, pancreas, lungs, liver, oesophagus, stomach, mammary gland, kidney especially originating from people
Dirty, gall-bladder, colon or rectum, ovary, uterus, cervix, the primary or secondary cancer and sarcoma of prostate, bladder, testis.
Claims (8)
1. a kind of method for extracting active component from centipede, the steps include:
A. centipede alcohol extract is prepared with the alcoholic solution that volumetric concentration is 45-65%；
B. using the centipede alcohol extract in Gel-filtration purification procedures a, multiple component samples is collected, this is detected
The anti-tumor activity of a little component samples, the best component of anti-tumor activity are denoted as component sample A, contain 3,8- in component sample A
Dihydroxy isoquinolin.
2. the method according to claim 1 for extracting active component from centipede, it is characterised in that: prepare Wu in step a
The step of centipede alcohol extract are as follows:
(1) Centipede is prepared, the PBS solution that 5-10 times of Centipede volume is then added carries out ultrasonication homogenate；
(2) to homogenate refrigerated centrifuge, supernatant is taken, the centipede water extract being lyophilized；
(3) alcoholic solution for taking the obtained water extract in step (2) that 5-10 times of Centipede volume is added is homogenized, and is then existed
It is leached under the conditions of 4 DEG C, the volumetric concentration of alcoholic solution is 45-65%；
(4) to leaching liquid refrigerated centrifuge again, supernatant is taken, the centipede alcohol extract being lyophilized.
3. the method according to claim 1 for extracting active component from centipede, it is characterised in that: use Portugal in step b
Polysaccharide gel chromatography isolates and purifies the step of centipede alcohol extract are as follows:
(1) sephadex Sephadex G25 is injected in chromatographic column, is rinsed with mobile phase, mobile phase is 10~50% second
Alcohol；
(2) centipede alcohol extract being configured to 20~50mg/mL, 4~8mL of loading in chromatographic column, flow velocity is 0.6~2mL/min,
Each component sample is collected according to chromatography map, Activity determination is carried out to each component sample, the best component of anti-tumor activity is denoted as
Component sample A.
4. a kind of method for preparing 3,8- dihydroxy isoquinolin, the steps include:
(a) centipede alcohol extract is prepared with the alcoholic solution that volumetric concentration is 45-65%；
(b) using the centipede alcohol extract in Gel-filtration purification procedures (a), multiple component samples are collected, are examined
The anti-tumor activity of these component samples is surveyed, the best component of anti-tumor activity is denoted as component sample A；
(c) using component sample A obtained in preparative RP-HPLC purification procedures (b), it is multiple to carry out gradient elution collection
Peak Activity component, detects the anti-tumor activity of these Peak Activity components, and the best component of anti-tumor activity is denoted as component B；
(d) it using the component B in semi-preparative RP-HPLC purification procedures (c), carries out gradient elution and collects multiple Peak Activities
Component, detects the anti-tumor activity of these Peak Activity components, and the best component of anti-tumor activity is 3,8- dihydroxy isoquinolin.
5. the active component extracted in claim 1 is in preparing anti-malaria medicaments, antibacterials and anti-tumor drug
Using.
6. active component according to claim 5 is preparing answering in anti-malaria medicaments, antibacterials and anti-tumor drug
With, it is characterised in that: the malaria is originating from the Plasmodium vivax, malariae, plasmodium falciparum for parasitizing human body
And Plasmodium ovale.
7. active component according to claim 5 is preparing answering in anti-malaria medicaments, antibacterials and anti-tumor drug
With, it is characterised in that: the bacterium is Escherichia coli, staphylococcus aureus, pseudomonas aeruginosa, first/hemolytic chain
Coccus, streptococcus pneumonia or bloodthirsty Bacillus influenzae.
8. active component according to claim 5 is preparing answering in anti-malaria medicaments, antibacterials and anti-tumor drug
With, it is characterised in that: the tumour be incidence originating from people, brain, thyroid gland, pancreas, lungs, liver, oesophagus, stomach,
Mammary gland, kidney, gall-bladder, colon or rectum, ovary, uterus, cervix, prostate, bladder, testis primary or secondary cancer with
And sarcoma.
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| CN101868447A (en) * | 2007-08-21 | 2010-10-20 | 香港理工大学 | Methods of making and administering quinoline derivatives as anticancer agents |
| CN102133233A (en) * | 2011-03-03 | 2011-07-27 | 湖南中医药大学 | Centipede extract capable of resisting tumor activity and preparation method thereof |
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