CN103553892B - Diterpene-kind compound and based on antineoplastic application - Google Patents
Diterpene-kind compound and based on antineoplastic application Download PDFInfo
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Abstract
The invention discloses two kinds of new diterpene-kind compounds and based on antineoplastic application.The invention provides compound shown in compound shown in formula (I) or formula (II).Does is Compound nomenclature Compound C yafrin shown in formula (I)? D.Does is Compound nomenclature Compound C yafrin shown in formula (II)? E.Compound C yafrin? D and Compound C yafrin? E all shows significant lethal effect to various tumor cell strains, and effectively can suppress human colon cancer cell HCT116 and Bax protein delation human colon cancer cell bax
-/-the growth of HCT116 in nude mouse.Bax protein on cells apoptosis plays a driving role, and its disappearance or mutant inactive often tumour cell produce one of reason of resistance.The present invention is that the new antitumor drug of development provides lead compound, has great value for oncotherapy.
Description
Technical field
The present invention relates to two kinds of new diterpene-kind compounds and based on antineoplastic application, particularly from cyathus extraction and isolation to two kinds of new Cyathane compounds and preparing the application in antitumor drug.
Background technology
Tumour is one of disease of the human health of serious harm.Tumour results from physiology cell proliferation out of control and have impact on the normal physiological conditions of human body, thus creates serious pathologic reaction, often causes death.In methods for the treatment of numerous at present, chemotherapeutics can arrive each portion of whole body with blood circulation, so chemotherapy (comprising immunotherapy) plays not replaceable effect in primary carcinoma, secondary metastatic carcinoma and leukemic treatment.It is single-minded that desirable chemotherapeutics should have action target spot, body internal specific target tumor tissue, and mechanism of action is clear and definite, the features such as normal tissue toxic side effect is little.The development of a large amount of I class original new drug (monomeric compound medicine) facts have proved: its pharmacological action of the structures shape of compound, new chemical structure probably has stronger biological activity, lower toxic side effect, is more suitable for Clinical practice.Therefore, an important directions in tumor research is exactly that find can single-minded blocking-up or kill tumour cell and do not affect the method for normal cell proliferation, is exactly find new antitumor drug in brief.
Summary of the invention
The object of this invention is to provide diterpene-kind compound and based on antineoplastic application.
The invention provides compound shown in compound shown in formula (I) or formula (II);
Shown in formula (I), Compound nomenclature is Compound C yafrinD.
Shown in formula (II), Compound nomenclature is Compound C yafrinE.
Present invention also offers the application of compound in the product preparing inhibition tumor cell propagation shown in compound shown in formula (I) or formula (II).Described tumour cell can be breast cancer cell, cervical cancer cell, colon cancer cell, liver cancer cell, human leukemia cell, Human Prostate Cancer Cells, human lung carcinoma cell or gastric carcinoma cells.Described tumour cell specifically can be MCF7 cell, HeLa cell, HCT116 cell, SW480 cell, HepG2 cell, K562 cell, PC3 cell, A549 cell or SGC-7901 cell.
The product that the present invention also protects a kind of inhibition tumor cell to breed, its activeconstituents is compound shown in formula (I) or formula (I) shown compound pharmacy acceptable salt, ester or solvent thing.Described tumour cell can be breast cancer cell, cervical cancer cell, colon cancer cell, liver cancer cell, leukemia cell, prostate cancer cell, lung carcinoma cell or stomach cancer cell.Described tumour cell specifically can be MCF7 cell, HeLa cell, HCT116 cell, SW480 cell, HepG2 cell, K562 cell, PC3 cell, A549 cell or SGC-7901 cell.
The product that the present invention also protects a kind of inhibition tumor cell to breed, its activeconstituents is compound shown in formula (II) or formula (II) shown compound pharmacy acceptable salt, ester or solvent thing.Described tumour cell can be breast cancer cell, cervical cancer cell, colon cancer cell, liver cancer cell, leukemia cell, prostate cancer cell, lung carcinoma cell or stomach cancer cell.Described tumour cell specifically can be MCF7 cell, HeLa cell, HCT116 cell, SW480 cell, HepG2 cell, K562 cell, PC3 cell, A549 cell or SGC-7901 cell.
The present invention goes back compound shown in compound shown in protection (I) or formula (II) and prepares the application treated and/or prevented in the product of tumour in preparation.Described tumour can be mammary cancer, cervical cancer, colorectal carcinoma, liver cancer, leukemia, prostate cancer, lung cancer or cancer of the stomach.Described tumour specifically can be the tumour that MCF7 cell, HeLa cell, HCT116 cell, SW480 cell, HepG2 cell, K562 cell, PC3 cell, A549 cell or SGC-7901 cell cause.
The present invention also protects a kind of product treating and/or preventing tumour, and its activeconstituents is compound shown in formula (I) or formula (I) shown compound pharmacy acceptable salt, ester or solvent thing.Described tumour can be mammary cancer, cervical cancer, colorectal carcinoma, liver cancer, leukemia, prostate cancer, lung cancer or cancer of the stomach.Described tumour specifically can be the tumour that MCF7 cell, HeLa cell, HCT116 cell, SW480 cell, HepG2 cell, K562 cell, PC3 cell, A549 cell or SGC-7901 cell cause.
The present invention also protects a kind of product treating and/or preventing tumour, and its activeconstituents is compound shown in formula (II) or formula (II) shown compound pharmacy acceptable salt, ester or solvent thing.Described tumour can be mammary cancer, cervical cancer, colorectal carcinoma, liver cancer, leukemia, prostate cancer, lung cancer or cancer of the stomach.Described tumour specifically can be the tumour that MCF7 cell, HeLa cell, HCT116 cell, SW480 cell, HepG2 cell, K562 cell, PC3 cell, A549 cell or SGC-7901 cell cause.
The present invention goes back the preparation method of compound shown in protection (I), comprises the steps: fermentation culture C.africanus, obtains compound shown in formula (I).Described C.africanus specifically can be C.africanus LH801.Described fermentation specifically can be solid fermentation.The condition of described solid fermentation specifically can be 22-28 DEG C of (as 25 DEG C) darkroom quiescent culture 10-50 days (as 38 days).The substratum (being called for short solid fermentation substratum) that described solid fermentation adopts can be made up of 1 weight part base material and 1-1.5 weight parts water.The substratum that described solid fermentation adopts can be made up of 1 weight part base material, 1-1.5 weight parts water and carbohydrate.Described base material is at least one in wheat, oat, polished rice, corn, glutinous rice, Gorgon fruit, long-grained nonglutinous rice and the seed of Job's tears.Described carbohydrate can be glucose.The substratum that described solid fermentation adopts specifically can be following substratum first, substratum second and substratum third.Described substratum first is made up of long-grained nonglutinous rice and water, and proportioning is 80g long-grained nonglutinous rice: 100ml water.Described substratum second is made up of wheat and water, and proportioning is 80g wheat: 90ml water.Described substratum third is made up of Gorgon fruit, glucose and water, and proportioning is 80g Gorgon fruit: 5g glucose: 90ml water.The step from compound extraction purification formula (I) the fermented product completing described solid fermentation Suo Shi is also comprised in described preparation method.Described extraction purification comprises the step of organic solvent extraction and chromatography successively.In described organic solvent extraction, described organic solvent specifically can be ethyl acetate.The step of described organic solvent extraction is specific as follows: get described fermented product, with the lixiviate of ethyl acetate normal temperature (can carry out a supersound process every 8 hours, the parameter of each supersound process specifically can be " 50HZ, 20min ").Described chromatography can comprise open column chromatography column purification (moving phase specifically can be chloroform-acetone) of silica gel and gel column sephadexLH-20 purifying (moving phase specifically can be chloroform-methanol).
The present invention goes back the preparation method of compound shown in protection (II), comprises the steps: fermentation culture C.africanus, obtains compound shown in formula (II).Described C.africanus specifically can be C.africanus LH801.Described fermentation specifically can be solid fermentation.The condition of described solid fermentation specifically can be 22-28 DEG C of (as 25 DEG C) darkroom quiescent culture 10-50 days (as 38 days).The substratum (being called for short solid fermentation substratum) that described solid fermentation adopts can be made up of 1 weight part base material and 1-1.5 weight parts water.The substratum that described solid fermentation adopts can be made up of 1 weight part base material, 1-1.5 weight parts water and carbohydrate.Described base material is at least one in wheat, oat, polished rice, corn, glutinous rice, Gorgon fruit, long-grained nonglutinous rice and the seed of Job's tears.Described carbohydrate can be glucose.The substratum that described solid fermentation adopts specifically can be following substratum first, substratum second and substratum third.Described substratum first is made up of long-grained nonglutinous rice and water, and proportioning is 80g long-grained nonglutinous rice: 100ml water.Described substratum second is made up of wheat and water, and proportioning is 80g wheat: 90ml water.Described substratum third is made up of Gorgon fruit, glucose and water, and proportioning is 80g Gorgon fruit: 5g glucose: 90ml water.The step from compound extraction purification formula (I) the fermented product completing described solid fermentation Suo Shi is also comprised in described preparation method.Described extraction purification comprises successively by organic solvent extraction and the step with chromatography.In described organic solvent extraction, described organic solvent specifically can be ethyl acetate.The step of described organic solvent extraction is specific as follows: get described fermented product, with the lixiviate of ethyl acetate normal temperature (can carry out a supersound process every 8 hours, the parameter of each supersound process specifically can be " 50HZ, 20min ").Described chromatography can comprise open column chromatography column purification (moving phase specifically can be chloroform-acetone) of silica gel and the reverse silica gel purification of ODS (moving phase specifically can be methanol-water).
C.africanus (Cyathusafricanus) LH801 is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on October 28th, 2013 and (is called for short CGMCC, address is: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica), deposit number is CGMCCNO.8411.
Compound C yafrinD and Compound C yafrinE all shows significant lethal effect to various tumor cell strains, and effectively can suppress human colon cancer cell HCT116 and Bax protein delation human colon cancer cell bax
-/-the growth of HCT116 in nude mouse.Bax protein on cells apoptosis plays a driving role, and its disappearance or mutant inactive often tumour cell produce one of reason of resistance.The present invention is that the new antitumor drug of development provides lead compound, has great value for oncotherapy.
Accompanying drawing explanation
Fig. 1 is the crystalline structure of Compound C yafrinD.
Fig. 2 be Compound C yafrinE with [Rh
2(OCOCF
3)
4] formed mixture CD difference spectrum.
Fig. 3 is the high-efficiency liquid-phase fingerprint in embodiment 2
Fig. 4 is the follow-up analysis figure of the fermented incubation time in embodiment 2.
Fig. 5 is in embodiment 3, tests the 24th day, the photo of nude mice.
Fig. 6 is in embodiment 3, and test the 24th day, the body weight of nude mice is shown in Fig. 6.
Fig. 7 is in embodiment 3, and test the 24th day, Fig. 7 is shown in by the photo cutting the tumour of getting off.
Fig. 8 is in embodiment 3, tests the gross tumor volume of the 10th day, the 12nd day, the 14th day, the 16th day, the 18th day, the 20th day, the 22nd day and the 24th day.
Fig. 9 is in embodiment 4, tests the 24th day, the photo of nude mice.
Figure 10 is in embodiment 4, and test the 24th day, the body weight of nude mice is shown in Fig. 6.
Figure 11 is in embodiment 4, and test the 24th day, Fig. 7 is shown in by the photo cutting the tumour of getting off.
Figure 12 is in embodiment 4, tests the gross tumor volume of the 10th day, the 12nd day, the 14th day, the 16th day, the 18th day, the 20th day, the 22nd day and the 24th day.
Embodiment
Following embodiment is convenient to understand the present invention better, but does not limit the present invention.Experimental technique in following embodiment, if no special instructions, is ordinary method.Test materials used in following embodiment, if no special instructions, is and purchases available from routine biochemistry reagent shop.Quantitative test in following examples, all arranges and repeats experiment for three times, results averaged.
The acquisition of embodiment 1, C.africanus LH801
C.africanus (Cyathusafricanus) LH801, is separated from Linzhi Area of Tibet Cyathusafricanus sporophore.
The basidioma of C.africanus LH801 is doline, high 5.5-8.0mm, oral area wide (9.0-11.0) × (6.5-8.0) μm; Wrap by without obvious vertical stripe; Parcel tool individual layer cortex and film, oblate, (1.9-2.5) × (1.8-2.0) mm; Sporidium is avette has a little point, (9.0-11.5) × (6.0-8.0) μm.
The sequence of the 18SrDNA of C.africanus LH801 is as shown in sequence in sequence table 1.
C.africanus LH801 is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on October 28th, 2013 and (is called for short CGMCC, address is: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica), deposit number is CGMCCNO.8411.
The preparation of embodiment 2, compound
One, the solid fermentation of C.africanus bacterium
1, bacterial strain activation
C.africanus LH801 is seeded to PDA culture medium flat plate, and 25 DEG C of lucifuges are cultivated 7 days (mycelia covers with whole flat board).
PDA substratum (natural pH): potato 200g, glucose 20g, agar powder 18g, be settled to 1L with pure water.
2, seed culture
Mycelium in step 1 is forwarded to liquid PDB substratum, 28 DEG C, 180rmp lucifuge cultivates 7 days, obtains seed culture fluid.
PDB substratum (natural pH): potato 200g, glucose 20g, pure water is settled to 1L.
3, solid fermentation is cultivated
Get the seed culture fluid that 5ml step 2 obtains, be seeded to solid fermentation substratum and (in each 500ml triangular flask, a solid fermentation substratum be housed, 10 triangular flasks are set, every part of solid fermentation substratum is made up of 80g long-grained nonglutinous rice and 100ml water), 25 DEG C of darkroom quiescent culture 38 days, obtain fermented product (the whole culture system that the material produced after namely being utilized by bacterium by the metabolite of thalline, bacterium, substratum and substratum forms).
Two, the preparation of compound
1, get the fermented product that step one obtains, add 300 milliliters of ethyl acetate normal temperature lixiviate 7 days (carried out a supersound process every 8 hours, the parameter of each supersound process is " 50HZ, 20min ") in each triangular flask, get supernatant liquor; Residuum adds 300 milliliters of new ethyl acetate normal temperature lixiviates 7 days (carried out a supersound process every 8 hours, the parameter of each supersound process is " 50HZ, 20min ") again, gets supernatant liquor; Residuum adds 300 milliliters of new ethyl acetate normal temperature lixiviates 7 days (carried out a supersound process every 8 hours, the parameter of each supersound process is " 50HZ, 20min "), gets supernatant liquor; The supernatant liquor three lixiviates obtained merges, and then vacuum distillation drying, obtains 13.6g medicinal extract (i.e. ethyl acetate extract).
2, get the 13.6g medicinal extract that step 1 obtains, dissolve with chloroform-methanol (volume ratio of chloroform and methyl alcohol is 1:1), the centrifugal 5min of 5000rpm, collect supernatant liquor, supernatant liquor is mixed with 13.6g column chromatography silica gel (Qingdao Marine Chemical Co., Ltd. 200-300 order), evaporate to dryness organic solvent in 50 DEG C of water-baths, then the open column chromatography post of silica gel (Φ 4.5 × 80cm is added into, be filled with 150g column chromatography silica gel, Qingdao Marine Chemical Co., Ltd. 200-300 order, column chromatography silica gel), chloroform-acetone is adopted to carry out gradient elution (in gradient elution process as moving phase again, volume ratio is adopted to be 100:0 successively, 100:1, 50:1, 30:1, 20:1, 10:1, 8:2, 7:3, chloroform-the acetone of 6:4 and 0:1, the elution volume of each gradient is 1500 milliliters), collect the front 1000 milliliter elutriants (solution first) of chloroform-acetone as moving phase of employing 50:1, collect and adopt the chloroform-acetone of 20:1 as rear 500 milliliters of elutriants of moving phase and the mixed solution (solution second) of chloroform-acetone as front 500 milliliters of elutriants of moving phase adopting 10:1.
3, by solution first concentrating under reduced pressure evaporate to dryness, dry-matter first (0.82g) is obtained.
4, the dry-matter first that 0.82g step 3 obtains is got, dissolve with chloroform-methanol (volume ratio of chloroform and methyl alcohol is 1:1), the centrifugal 5min of 5000rpm, collect supernatant liquor, supernatant liquor is splined on gel column sephadexLH-20(Φ 2.5 × 120cm, sephadexLH-20, Merck company), then adopt chloroform-methanol (volume ratio of chloroform and methyl alcohol is 1:1) to carry out wash-out as moving phase, collect the elutriant (solution third) of the 120th milliliter to the 180th milliliter.
5, by solution third concentrating under reduced pressure evaporate to dryness, dry-matter third (0.51g) is obtained.
6, dry-matter third is single sterling compound, by its called after Compound C yafrinD.
CyafrinD is clear crystal, and molecular formula positive ion mode HR-ESI-MS shows m/z355.2240 [M+Na]
+(355.2244, calculated value), determine that its molecular formula is C
21h
32o
3, calculating its degree of unsaturation (Ω) is 6.
Compound C yafrinD's
1h-NMR and
13c-NMR spectrogram shows, it exists an aldehyde radical, a methoxyl group and an isopropyl group.The two dimensional structure of Compound C yafrinD can be drawn in conjunction with HMBC collection of illustrative plates.The nuclear-magnetism ownership of Compound C yafrinD, in table 1, is new compound through SciFinderScholar literature search.
The nuclear-magnetism ownership of table 1 Compound C yafrinD
a1HNMRat500MHz,
13CNMRat125MHzinCD
3OD.
breference literature Fitoterapia, 2013,84,22-31.
* J is coupling constant, unit Hz.
The relative configuration of Compound C yafrinD is that the long-range coherent signal of H-2 and H-3 by observing in NOESY collection of illustrative plates and H-9 is determined.H3-17 has coherent signal with H-5, H-8 β and H-1 β respectively, and H3-16 has coherent signal with H-7 α and H-14 respectively, therefore can infer the relative configuration compound.By obtaining the crystalline structure (see figure 1) of Compound C yafrinD by X single crystal diffraction technology (copper target test compounds), not only confirm its two dimensional structure, also determine its relative configuration, concurrent present 11-OCH
3as Compound C yathinE, H key in a molecule is not formed with between 14-OH.The Flack constant of measuring is-0.05 (16), according to crystal rule (ActaCryst., 1983, A39:876-881), determines that its absolute configuration is 5R, 6R, 9R, 11R and 14S.
Compound C yafrinD structural formula is as follows:
7, by solution second concentrating under reduced pressure evaporate to dryness, dry-matter second (1.51g) is obtained.
8, get the dry-matter second that 1.51g step 7 obtains, dissolve with chloroform-methanol (volume ratio of chloroform and methyl alcohol is 1:1), the centrifugal 5min of 5000rpm, collect supernatant liquor, by supernatant liquor and the reverse silica gel (YMC*GEL of 1.51gODS, 12nm, S-50um) mix, evaporate to dryness organic solvent in 50 DEG C of water-baths, then (Φ 3.5 × 50cm in mesolow chromatographic column is added into, be filled with the anti-phase ODS silica gel of 80g, YMC*GEL, 12nm, S-50um), then methanol-water is adopted to carry out gradient elution (in gradient elution process as moving phase, volume ratio is adopted to be 20:80 successively, 35:65, 45:55, 60:40, 70:30, 80:20, the methanol-water of 90:10 and 0:1, the elution volume of each gradient is 300 milliliters), collect the rear 200ml elutriant (solution fourth) of methanol-water as moving phase of employing 70:30.
9, by solution fourth concentrating under reduced pressure evaporate to dryness, dry-matter fourth (78mg) is obtained.
10, dry-matter fourth is single sterling compound, by its called after Compound C yafrinE.
Compound C yafrinE is white crystal, and positive ion mode HR-ESI-MS shows m/z355.2239 [M+Na]
+(355.2244, calculated value), determine that its molecular formula is C
21h
32o
3, calculating its degree of unsaturation (Ω) is 6.
Compound C yafrinE's
1h-NMR and
13c-NMR spectrogram shows, it exists an aldehyde radical, a methoxyl group and an isopropyl group.In HMBC collection of illustrative plates, H
3-16(δ 1.10,3H, s) relevant to C-5, C-6, C-7 and C-14, H-21(δ 3.25,3H, s) relevant to C-13, H-13(δ 4.53, dd, J=5.9 & 1.1Hz) relevant to C-6, C-11, C-14 and C-15, H-14 and C-5, C-7, C-13, C-12 are relevant with C-16, and then determine the two dimensional structure of Compound C yafrinE.The nuclear-magnetism ownership of Compound C yafrinE, in table 2, is new compound through SciFinderScholar literature search.
The nuclear-magnetism ownership of table 2 Compound C yafrinE
a1HNMRat500MHz,
13CNMRat125MHzinCD
3OD.
breference literature Biosci.Biotechnol.Biochem., 2004,68:1362-1365.
* J is coupling constant, unit Hz.
The relative configuration of Compound C yafrinE is determined by NOESY.According to H-13 and H-14 coupling constant J=5.9Hz, infer that they may be cis-structure.Also can find from NOESY spectrogram, H-5 respectively with H
3-17, H-13 and H-10 β has relevant, H
3-16 have relevant to H-14 and H-10 α, can determine the relative configuration of this compound thus, demonstrate supposition above.In addition according to H-13(δ 4.53, dd, J=1.1 & 5.9Hz), and in known compound neosarcodoninO, being one, unimodal (δ 4.62 s), also illustrates between H-13 and H-14 to be cis-structure.By analysis of compounds 14-OH and [Rh
2(OCOCF
3)
4] form the CD feature of complex compound and also determine its absolute configuration.As the hydroxyl in compound and [Rh
2(OCOCF
3)
4] formed mixture time; its CD composes in E band place (about 350nm) place display Cotton effect, according to " bulkiness " rule, and the positive and negative absolute configuration (Tetrahedron:Asymmetry that indirectly can reflect the carbon connecting hydroxyl of this Cotton effect; 1999,10:1507-1520; Tetrahedron:Asymmetry, 1990,1:221-236).Compound C yafrinE and [Rh
2(OCOCF
3)
4] in the CD spectrogram of mixture that formed (see Fig. 2; The CD spectrum of compound itself is deducted), show negative Cotton effect at 350nm place, the steric configuration determining 14 is 14R, and in conjunction with the relative configuration determined by NOESY data, thus to determine its absolute configuration be 5R, 6R, 9R, 13R and 14R.
The structural formula of Compound C yafrinE is as follows:
Embodiment 2, different fermentation parameters is adopted to prepare compound
One, solid fermentation scheme one
1, solid fermentation
With the step one of embodiment 1.
2, the detection of fermented product
Get the fermented product that step 1 obtains, lixiviate 21 day (the every ethyl acetate that 7 day more renew ultrasonic with ethyl acetate normal temperature; Carried out a supersound process every 8 hours, the parameter of each supersound process is " 50HZ, 20min "), the supernatant liquor three lixiviates obtained merges, and then vacuum distillation drying, obtains medicinal extract; Use dissolve with methanol medicinal extract, obtain the solution of 10mg/mL, carry out high performance liquid chromatography detection.
Colleges and universities' liquid phase chemical fingerprint map analyzing condition is as follows: use Agilent Agilent1200 high performance liquid chromatograph, quaternary gradient pump, DAD detector, Agilent chromatographic working station; Chromatographic column is AgilentC18 analytical column (4.6mm × 150mm, 5 μm); Elution program is in table 3, and flow velocity is 1.00mL/min; Ultraviolet detection wavelength 220nm.
Table 3 elution program (linear gradient elution)
High-efficient liquid phase chromatogram is shown in Fig. 3 A.
Two, solid fermentation scheme two
1, solid fermentation
Adopt following solid fermentation substratum: be made up of 80g wheat and 90ml water.
Other is all with the step one of embodiment 1.
2, the detection of fermented product
With 2 of step one.
High-efficient liquid phase chromatogram is shown in Fig. 3 B.
Three, solid fermentation scheme three
1, solid fermentation
Adopt following solid fermentation substratum: be made up of 80g Gorgon fruit, 5g glucose and 90ml water.
Other is all with the step one of embodiment 1.
2, the detection of fermented product
With 2 of step one.
High-efficient liquid phase chromatogram is shown in Fig. 3 C.
Four, fermentation time
Solid fermentation is with the step one of embodiment 1.Respectively at the different time sampling of fermentation, according to the method detection fermented product of 2 of step one.
The results are shown in Figure 4.Between fermentation culture 35-40 days, better, output is the highest for Compound C yafrinD and Compound C yafrinE richness.
The anti-tumor activity of embodiment 3, Compound C yafrinD and Compound C yafrinE
One, cell experiment
Detection compound CyafrinD and Compound C yafrinE is to the inhibiting rate of various tumour cell respectively.Tumour cell is MCF7 cell (ATCCHTB-22; Breast cancer cell), HeLa cell (ATCCCCL-2; Cervical cancer cell), HCT116 cell (ATCCCCL-247; Colon cancer cell), SW480 cell (ATCCCCL-228; Colon cancer cell), HepG2 cell (ATCCHB-8065; Liver cancer cell), K562 cell (ATCCCCL-243; Human leukemia cell), PC3 cell (ATCCCRL-1435; Human Prostate Cancer Cells), A549 cell (ATCCCCL-185; Human lung carcinoma cell) or SGC-7901 cell (ATCCSGC-7901; Gastric carcinoma cells).HepG2 cell, K562 cell, PC3 cell adopt and cultivate containing the DMEM nutrient solution of 10% foetal calf serum.MCF7 cell, HeLa cell, HCT116 cell, SW480 cell, A549 cell and SGC-7901 cell adopt and cultivate containing the RPMI1640 nutrient solution of 10% foetal calf serum.Each cell is all at 37 DEG C, 5%CO
2cellar culture in incubator, goes down to posterity every other day.
1, get tumour cell, with trysinization, make 1 × 10
5individual cell/milliliter single cell suspension.
2, single cell suspension is inoculated in (every hole 200 μ L) in 96 orifice plates, Compound C yafrinD(or Compound C yafrinE is added after 24 hours) and the concentration range making it in culture system is 100 μMs to 1.56 μMs (diluting 6 gradients from 100 μMs of sesquialters), cultivates 48 hours; Arrange simultaneously and do not add Compound C yafrinD(or Compound C yafrinE) blank group and the 5-FU(5-fluorine that adds each concentration urinate phonetic) positive controls.Often kind of process arranges three multiple holes.
3, after completing steps 2, measure tumor cell number by mtt assay, calculate inhibiting rate (%).
Inhibiting rate (%)=(1-administration group OD value/blank group OD value) × 100%.
IC
50=[CL (IH-50)+CH (50-IL)]/(IH-IL); CL: lower concentration values; CH: high concentration value; IH: the inhibiting rate under high density; IL: the inhibiting rate under lower concentration.
Compound C yafrinD or Compound C yafrinE is to the IC of each tumour cell
50value (unit for μM) is in table 3.Compound C yafrinD or Compound C yafrinE all shows the rejection ability stronger to tumour cell.
Table 3 Compound C yafrinD or Compound C yafrinE is to the IC of each tumour cell
50value (unit be μM)
| HepG2 | K562 | PC3 | MCF7 | HeLa | HCT116 | SW480 | A549 | SGC-7901 | |
| Cyafrin D | 5.79 | 8.54 | 9.83 | 10.93 | 6.12 | 3.23 | 9.52 | 8.56 | 9.81 |
| Cyafrin E | 15.55 | 5.45 | 13.41 | 21.45 | 5.18 | 2.26 | 5.63 | 17.15 | 19.52 |
| 5-FU | 14.23 | 6.82 | 26.54 | 35.21 | 20.2 | 12.3 | 23.1 | 18.91 | 21.64 |
Two, experimentation on animals
HCT116 cell: Abcam company produces, Central Plains, Beijing corporate agent.
4-5 (20-24g, Chinese Academy of Medical Sciences's Beijing Institute of Botany) 60 in SPF level BALB/c Female nude mice in age in week, tests the 1st day, and every nude mice dorsal sc inoculation HCT116 cell 0.2ml(contains 2 × 10
6individual logarithmic phase cell), test the 10th day knurl block and be shaped (diameter reaches 0.5-1.0cm), nude mice is divided at random 5 groups (often organizing 12), is handled as follows respectively:
First group: respectively inject 0.2ml Compound C yafrinD solution respectively at experiment the 11st day, the 13rd day, the 15th day, the 17th day, the 19th day, the 21st day and the 23rd day abdominal cavity and (be dissolved in DMSO by Compound C yafrinD, again with the dilution of PBS damping fluid), the dosage that each abdominal injection gives Compound C yafrinD is 10mg/kg body weight;
Second group: respectively inject 0.2ml Compound C yafrinD solution respectively at experiment the 11st day, the 13rd day, the 15th day, the 17th day, the 19th day, the 21st day and the 23rd day abdominal cavity and (be dissolved in DMSO by Compound C yafrinD, again with the dilution of PBS damping fluid), the dosage that each abdominal injection gives Compound C yafrinD is 5mg/kg body weight;
3rd group: respectively inject 0.2ml Compound C yafrinE solution respectively at experiment the 11st day, the 13rd day, the 15th day, the 17th day, the 19th day, the 21st day and the 23rd day abdominal cavity and (be dissolved in DMSO by Compound C yafrinE, again with the dilution of PBS damping fluid), the dosage that each abdominal injection gives Compound C yafrinE is 10mg/kg body weight;
4th group: respectively inject 0.2ml Compound C yafrinE solution respectively at experiment the 11st day, the 13rd day, the 15th day, the 17th day, the 19th day, the 21st day and the 23rd day abdominal cavity and (be dissolved in DMSO by Compound C yafrinE, again with the dilution of PBS damping fluid), the dosage that each abdominal injection gives Compound C yafrinE is 5mg/kg body weight;
5th group (control group): respectively inject 0.2mlPBS damping fluid respectively at experiment the 11st day, the 13rd day, the 15th day, the 17th day, the 19th day, the 21st day and the 23rd day abdominal cavity.
Respectively at experiment the 10th day, within the 12nd day, the 14th day, the 16th day, the 18th day, the 20th day, the 22nd day and the 24th day, measure tumor volume and nude mice body weight.Test the 24th day, put to death mouse, calculate gross tumor volume inhibiting rate (IR).
V is gross tumor volume, and a is tumour major diameter (mm), b is tumour minor axis (mm).
C is control group tumor average volume, and T is administration group tumor average volume.
Statistical study, adopt t inspection process, P<0.05 thinks significant difference.
Test the 24th day, Fig. 5 is shown in by the photo of nude mice, and the body weight of nude mice is shown in Fig. 6, and Fig. 7 is shown in by the photo cutting the tumour of getting off.The gross tumor volume of testing the 10th day, the 12nd day, the 14th day, the 16th day, the 18th day, the 20th day, the 22nd day and the 24th day is shown in Fig. 8.Test the mean tumor volume of the 24th day, IR the results are shown in Table 4.Compound C yafrinD and Compound C yafrinE all has obvious restraining effect to Human Colonic Tumor in Nude Mice transplanted tumor.Medication mouse and control mice body weight do not have notable difference, have no the difference of the aspects such as mouse outward appearance, active state.
The mean tumor volume of the 24th day tested by table 4, IR result
Three, experimentation on animals
Bax
-/-hCT116 cell: reference: AnaturalcompoundelevatesexpressionoftheBimthatinteractsw ithBcl-2convertinganti-apoptoticproteintoapro-apoptoticB ax-likemolecule(JBC, 2012, vol.287no.21054-1065.Use bax
-/-hCT116 cell replaces HCT116 cell, other same step 2.
Test the 24th day, Fig. 9 is shown in by the photo of nude mice, and the body weight of nude mice is shown in Figure 10, and Figure 11 is shown in by the photo cutting the tumour of getting off.The gross tumor volume of testing the 10th day, the 12nd day, the 14th day, the 16th day, the 18th day, the 20th day, the 22nd day and the 24th day is shown in Figure 12.Test the mean tumor volume of the 24th day, IR the results are shown in Table 5.Compound C yafrinD and Compound C yafrinE all has obvious restraining effect to the Human Colonic Tumor in Nude Mice transplanted tumor of Bax protein delation.Medication mouse and control mice body weight do not have notable difference, have no the difference of the aspects such as mouse outward appearance, active state.
The mean tumor volume of the 24th day tested by table 5, IR result
| Test the mean tumor volume (cm of the 24th day 3) | IR(%) | P value | |
| 5th group | 1.783216±0.132052 | - | |
| Second group | 0.99462±0.1321582 | 44.22325 | ＜0.01 |
| First group | 0.56041±0.06563512 | 68.57307 | ＜0.01 |
| 4th group | 1.355462±0.11582 | 23.98778 | ＜0.01 |
| 3rd group | 0.79041±0.06532635 | 55.67503 | ＜0.01 |
Claims (9)
1. compound shown in formula (I) or the shown compound of formula (II);
2. compound shown in the formula (I) in claim 1 or the application of the shown compound of formula (II) in the product preparing inhibition tumor cell propagation.
3. a product for inhibition tumor cell propagation, its activeconstituents is compound shown in the formula (I) in claim 1 or the shown compound pharmacy acceptable salt of formula (I).
4. a product for inhibition tumor cell propagation, its activeconstituents is compound shown in the formula (II) in claim 1 or the shown compound pharmacy acceptable salt of formula (II).
5. compound shown in the formula (I) in claim 1 or the shown compound of formula (II) are preparing the application treated and/or prevented in the product of tumour.
6. treat and/or prevent a product for tumour, its activeconstituents is compound shown in the formula (I) in claim 1 or the shown compound pharmacy acceptable salt of formula (I).
7. treat and/or prevent a product for tumour, its activeconstituents is compound shown in the formula (II) in claim 1 or the shown compound pharmacy acceptable salt of formula (II).
8. the preparation method of compound shown in formula described in claim 1 (I), comprises the steps: fermentation culture C.africanus, obtains compound shown in formula (I); Described C.africanus is C.africanus LH801; Described fermentation is solid fermentation; The condition of described solid fermentation is 22-28 DEG C of darkroom quiescent culture 10-50 days; The substratum that described solid fermentation adopts can be made up of 1 weight part base material, 1-1.5 weight parts water and carbohydrate; Described base material is at least one in wheat, oat, polished rice, corn, glutinous rice, Gorgon fruit, long-grained nonglutinous rice and the seed of Job's tears; Described carbohydrate can be glucose; The substratum that described solid fermentation adopts is following substratum first, substratum second and substratum third; Described substratum first is made up of long-grained nonglutinous rice and water, and proportioning is 80g long-grained nonglutinous rice: 100ml water; Described substratum second is made up of wheat and water, and proportioning is 80g wheat: 90ml water; Described substratum third is made up of Gorgon fruit, glucose and water, and proportioning is 80g Gorgon fruit: 5g glucose: 90ml water; The step from compound extraction purification formula (I) the fermented product completing described solid fermentation Suo Shi is also comprised in described preparation method; Described extraction purification comprises the step of organic solvent extraction and chromatography successively; In described organic solvent extraction, described organic solvent is ethyl acetate; The step of described organic solvent extraction is as follows: get described fermented product, with the lixiviate of ethyl acetate normal temperature, carries out a supersound process every 8 hours, and the parameter of each supersound process is 50HZ, 20min; Described chromatography comprises the open column chromatography column purification of silica gel and gel column sephadexLH-20 purifying; Described silica gel open column chromatography column purification moving phase is chloroform-acetone; Described gel column sephadexLH-20 purifying moving phase is chloroform-methanol.
9. the preparation method of compound shown in formula described in claim 1 (II), comprises the steps: fermentation culture C.africanus, obtains compound shown in formula (II); Described C.africanus is C.africanus LH801; Described fermentation is solid fermentation; The condition of described solid fermentation is 22-28 DEG C of darkroom quiescent culture 10-50 days; The substratum that described solid fermentation adopts can be made up of 1 weight part base material, 1-1.5 weight parts water and carbohydrate; Described base material is at least one in wheat, oat, polished rice, corn, glutinous rice, Gorgon fruit, long-grained nonglutinous rice and the seed of Job's tears; Described carbohydrate can be glucose; The substratum that described solid fermentation adopts is following substratum first, substratum second and substratum third; Described substratum first is made up of long-grained nonglutinous rice and water, and proportioning is 80g long-grained nonglutinous rice: 100ml water; Described substratum second is made up of wheat and water, and proportioning is 80g wheat: 90ml water; Described substratum third is made up of Gorgon fruit, glucose and water, and proportioning is 80g Gorgon fruit: 5g glucose: 90ml water; The step from compound extraction purification formula (I) the fermented product completing described solid fermentation Suo Shi is also comprised in described preparation method; Described extraction purification comprises by organic solvent extraction and the step with chromatography successively; In described organic solvent extraction, described organic solvent is ethyl acetate; The step of described organic solvent extraction is as follows: get described fermented product, with the lixiviate of ethyl acetate normal temperature, carries out a supersound process every 8 hours, and the parameter of each supersound process is 50HZ, 20min.Described chromatography comprises the open column chromatography column purification of silica gel and the reverse silica gel purification of ODS; The open column chromatography column purification flowing of described silica gel is chloroform-acetone; The reverse silica gel purification moving phase of described ODS is methanol-water.
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| WO2007123027A1 (en) * | 2006-04-10 | 2007-11-01 | Geol Chemical Co., Ltd. | Anti-cancer agent comprising cyathane derivative |
| CN102846588A (en) * | 2012-09-20 | 2013-01-02 | 中国科学院微生物研究所 | Application of cyathane diterpene compound in antitumor drug |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2007123027A1 (en) * | 2006-04-10 | 2007-11-01 | Geol Chemical Co., Ltd. | Anti-cancer agent comprising cyathane derivative |
| CN102846588A (en) * | 2012-09-20 | 2013-01-02 | 中国科学院微生物研究所 | Application of cyathane diterpene compound in antitumor drug |
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| Anti-inflammatory and cytotoxic cyathane diterpenoids from the medicinal fungus Cyathus africanus;JunJie Han;《Fitoterapia》;20121214;第84卷;第22-31页 * |
| Isolation and identification of a new anti-inflammatory cyathane diterpenoid from the medicinal fungus Cyathus hookeri Berk;Zhenyu Xu;《Fitoterapia》;20130314;第86卷;第159-162页 * |
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