CN105055395A - Application of Cephaloziellin B in preparation of medicine for treating ovarian cancer - Google Patents
Application of Cephaloziellin B in preparation of medicine for treating ovarian cancer Download PDFInfo
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- CN105055395A CN105055395A CN201510578917.4A CN201510578917A CN105055395A CN 105055395 A CN105055395 A CN 105055395A CN 201510578917 A CN201510578917 A CN 201510578917A CN 105055395 A CN105055395 A CN 105055395A
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- cephaloziellinb
- ovarian cancer
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Abstract
The invention discloses application of Cephaloziellin B in the preparation of a medicine for treating an ovarian cancer, and belongs to the field of medicines. A research shows that the Cephaloziellin B has an inhibition effect on the proliferation of SKOV3 cells of the ovarian cancer, the inhibition rate is concentration-dependent and time-dependent, and the Cephaloziellin B can be further researched and developed to prepare the medicine for treating the ovarian cancer.
Description
Technical field
The present invention relates to the novelty teabag of Compound C ephaloziellinB, be specifically related to the application of CephaloziellinB in preparation treatment ovarian cancer.
Background technology
The people such as Rui-JuanLi separation and purification first goes out Compound C ephaloziellinB, and achievement is published in famous natural product magazine (SecondaryMetabolitesfromtheChineseLiverwortCephaloziella kiaeri, J.Nat.Prod., 2013,76,1700-1708).
Not yet there is this compound at present about the active reporter for the treatment of ovarian cancer.
Summary of the invention
The object of the present invention is to provide the medical usage of a kind of CephaloziellinB.
Above-mentioned purpose is achieved by the following technical solution:
The application of CephaloziellinB in preparation treatment ovarian cancer, described CephaloziellinB chemical structural formula is as follows,
Further, described ovarian cancer is SKOV3.
Detailed description of the invention
Essentiality content of the present invention is further illustrated below in conjunction with embodiment.
The separation preparation of embodiment 1:CephaloziellinB and structural identification
The preparation method of CephaloziellinB is with the preparation method (BioactiveLimonoidandTriterpenoidConstituentsofTurraeapub escens, J.Nat.Prod., 2013,76,1166-1174) of bibliographical information.
Structural identification: white crystals (methanol), fusing point 156-158 DEG C.Be C according to the known molecular formula of HR-ESI-MS
20h
22o
5, degree of unsaturation is 10.Hydrogen nuclear magnetic resonance modal data δ
h(ppm, DMSO-d
6, 600MHz): H-1 (2.07, m), H-1 (1.89, dd, J=15.6, 7.2), H-2 (2.42, m), H-2 (2.58, m), H-3 (6.91, br, s), H-6 (4.59, dd, J=11.4, 7.8), H-7 (2.28, dd, J=12.5, 7.8), H-7 (1.80, dd, J=12.0), H-10 (1.98, m), H-11 (3.01, dd, J=13.2, 7.8), H-11 (2.00, m), H-12 (5.30, t, J=8.4), H-14 (6.47, br, s), H-15 (7.35, br, s), H-16 (7.46, br, s), H-17 (1.20, s), H-19 (1.31, s), H-20 (5.50, s), carbon-13 nmr spectra data δ
c(ppm, DMSO-d
6, 600Hz): 19.1 (CH
2, 1-C), 24.0 (CH
2, 2-C), 138.4 (CH, 3-C), 132.9 (C, 4-C), 40.1 (C, 5-C), 84.7 (CH, 6-C), 42.0 (CH
2, 7-C), 72.6 (C, 8-C), 60.5 (C, 9-C), 45.9 (CH, 10-C), 38.0 (CH
2, 11-C), 74.7 (CH, 12-C), 128.9 (C, 13-C), 109.1 (CH, 14-C), 143.6 (CH, 15-C), 139.8 (CH, 16-C), 24.6 (CH
3, 17-C), 169.8 (C, 18-C), 30.1 (CH
3, 19-C), 101.0 (CH, 20-C).Structural identification data are consistent with bibliographical information, therefore can determine that compound prepared by the present invention is the CephaloziellinB of bibliographical information.
The pharmacological action test of embodiment 2:CephaloziellinB
One, material and instrument
People's serous papillary cystenoma of ovary shape cystadenocarcinoma Cell line SKOV3 is provided by the Medical experimental center of Lanzhou University.General RPMI-1640 culture medium (10% hyclone) is cultivated.CephaloziellinB makes by oneself, and HPLC normalization purity is greater than 98%.RPMI-1640 culture fluid, tetramethyl azo frustrate indigo plant (MTT), dimethyl sulfoxide (DMSO), L-glutaminate are purchased from Ke Hao biological engineering company limited.Trypsin is purchased from Sigma Co., USA.Hyclone is purchased from Hangzhou Sijiqing Biological Engineering Material Co., Ltd..Ten Second Academys base sodium sulfonate (SDS) are purchased from Xi'an Zhou Ding biotechnology Co., Ltd.HER2 (FITC labelling) is purchased from Beijing Bo Aosen Bioisystech Co., Ltd.Acidify HER2 (FITC labelling) is purchased from Beijing Bo Aosen Bioisystech Co., Ltd.Penicillin, streptomycin are purchased from North China Pharmaceutical Factory.
CO
2incubator (Heraeus, BB5060UV), Germany inverted phase contrast microscope (OLYMPUS, CK-40), Japan fluorescence microscope (OLYMPUSOPTICAL, AX80, Japan), superclean bench (Purifying Equipment Co., Ltd., Suzhou), enzyme micro-plate reader (BIO-TEK company, the U.S.), low-temperature and high-speed centrifuge (Beckman-Coulter company, Germany), EpicsXL flow cytometer (Beckman-Coulter company, Germany), the automatic desk-top fire extinguisher (Xinhua Medical Apparatus Co., Ltd. Shandong) of R-3850 type, DHG-9245A type electric heating constant-temperature blowing drying box (Shanghai-permanent Science and Technology Ltd.), KQ-250DB type numerical control ultrasonic cleaner (Kunshan Ultrasonic Instruments Co., Ltd.), digital display constant water bath box (Shanghai Mei Xiang Instrument Ltd.), BS400S electronic molecules balance (Beijing Sai Duolisi balance company limited), 08-2 constant temperature blender with magnetic force (Shanghai balance equipment factory), TDL-5 generic centrifuge (Anting Scientific Instrument Factory, Shanghai), cryogenic refrigerator (SANYO GS company), electronics ice making case (SANYO GS company), cell cryopreservation tube (Shanghai Sheng Gong biological engineering company limited), 96 orifice plates (Ke Hao biological engineering company limited).
Two, test method
1, cell culture
1.1 cell recovery
Put into 37 DEG C of warm water immediately after being taken out from liquid nitrogen rapidly by cryopreservation tube, rock cryopreservation tube gently, frozen thing is dissolved as early as possible, put it in superclean bench, cell suspension in it is moved into centrifuge tube, then in centrifuge tube, adds 10 times of RPMI-1640 culture fluid (containing 10% calf serum), centrifugal, 800rpm/min, centrifugal 5 ~ 10min, abandons supernatant, adds the RPMI-1640 culture fluid containing 10% calf serum in cell precipitation, jog is even, puts 37 DEG C, 5%CO
2cultivate in the incubator of concentration.And indicate Cell Name and date.
1.2 passages are cultivated
When SKOV3 cell grows to 80 ~ 90% culture bottle, discard original culture fluid, and wash twice with the PBS prepared; With the trypsinization of 0.25%, observe under being placed on inverted microscope, when seeing that cell retraction, intercellular substance increase, form become bowlder and discard Digestive system, add in a certain amount of culture fluid and trypsinization liquid, and the cell digested is blown and beaten gently with suction pipe., make it depart from culture bottle, the centrifugal 5min of 800rpm/min, abandons supernatant; On counting chamber, carry out cell counting under microscope, go down to posterity in 1:3 ratio, divide and be filled in culture bottle, continue to cultivate after again supplementing appropriate culture fluid.Note strict aseptic technique, every day observation of cell growing state.
1.3 cell cryopreservation
Take the logarithm the cell of trophophase with centrifugal after the trypsinization of 0.25%, PBS washes 2 times, the centrifugal 5min of l000rpm on low speed centrifuge, abandon supernatant, add the cells frozen storing liquid containing DMSO of 1mL pre-cooling at-20 DEG C, moving in cryopreservation tube with after suction pipe piping and druming evenly, with putting into 4 DEG C of standing 30min after sealed membrane sealing mark, proceeding to-80 DEG C after-20 DEG C of placement 2h afterwards.The cell used in one month can be stored in-80 DEG C, should move in liquid nitrogen after long-term preserver 24h by-80 DEG C.The recovery of cell and the frozen principle that should follow are melted soon for freezing slowly.
2, tetramethyl azo blue (MTT) experiment
(1), when selecting cell (the growth 80-90%) of exponential phase, with 0.25% pancreatin prepared, cell is disappeared.Change well, single cell suspension is made in piping and druming lightly, and the final cell concentration of adjustment is 8 × 10
4/ mL; (2) get 3 96 orifice plates, each time point 1 plate, 100 μ L/ holes, every porocyte number is 10
4carry out grouping mark; (3) divide into groups after cell attachment, if blank group (not inoculating cell), matched group (only containing equivalent solvent) and experimental group (add the CephaloziellinB of variable concentrations, its final concentration is respectively 0.1,1,10,20,30 and 60 μm of ol/L), often group establishes 6 multiple holes; (4) 37 DEG C, 5%CO
224,48 and 72h is cultivated respectively under condition; (5), after the time arrives, after sucking supernatant, every hole adds the MTT10 μ L of 5g/L; (6) add 10 μ LDMSO after cultivating 4h, multi-functional microplate test macro measures each hole optical density OD value with 490nm wavelength; (7) medicine is to the calculating of cell inhibitory rate:
Suppression ratio (%)=(cellular control unit number-experimental group cell number)/cellular control unit number × 100%.
Experiment at least in triplicate.
3, PI staining flow cytometry (FCM) detects cell cycle and apoptosis
(1) to take the logarithm the SKOV3 cell of trophophase, first with after the trypsinization prepared gently piping and druming make single cell suspension, adjustment cell concentration is 6 × 10
5/ mL; (2) with 6 × 10
5the density of/mL is inoculated in the culture bottle of 50mL, in 37 DEG C, 5%CO
224h is cultivated in incubator; (3) after 24h by original culture fluid sucking-off, add isodose (7.5mL), containing the culture fluid of the CephaloziellinB of variable concentrations (0,10,20 and 30 μm of ol/L), continue at 37 DEG C, 5%CO
248h is cultivated in incubator; (4) collecting cell after 48h, makes single cell suspension, after being moved into centrifuge tube with blowing and beating gently after 0.25% trypsinization, centrifugal with 1000r/min, centrifugal l0min, abandoning supernatant, and fix with the ice ethanol of 4 DEG C 70%, place the refrigerator overnight of 4 DEG C; (5) secondary daily phosphate buffer PBS clean, centrifugal, detect after abandoning supernatant and add ribonuclease (RNAase) 150 μ L and propidium iodide (PI) dye liquor 150 μ L, lucifuge dyeing 30min at ambient temperature, uses period profile and the apoptosis situation of flow cytomery cell.Test in triplicate, and application software is analyzed.
4, PI staining flow cytometry (FCM) detects the expression rate of HER2, P-HER2 albumen
(1) to take the logarithm the SKOV3 cell of trophophase, first with after 0.25% trypsinization gently piping and druming make single cell suspension, adjustment cell concentration is l × 10
6/ mL; (2) with l × 10
6the density of/mL is inoculated in the culture bottle of 50mL, in 37 DEG C, 5%CO
224h is cultivated in incubator; (3) the original culture fluid of sucking-off after 24h, the culture fluid (matched group) of the culture fluid (experimental group) of CephaloziellinB that add isodose (12.5mL), that containing concentration be 20 μm of ol/L and not drug containing, continues at 37 DEG C, 5%CO
248h is cultivated in incubator; (4) collecting cell after 48h, first moves into the culture fluid in culture bottle in centrifuge tube, then uses the trypsinization of 0.25%, and the cell in guarantee culture bottle and nutritional solution all move into centrifuge tube, centrifugal by 1000r/min, l0min; (5) add PBS again by the centrifugal 10min of 1000 turns/min, repeat twice; (6) treat in test tube, there are 100 μ L samples, add HER2, P-HER2 antibody receptor (dissolve with the PBS of PH=7.4,0.01M, and press 1:60 dilution) of 30 μ LFITC labellings to experimental group and matched group respectively, hatch 30min for 4 DEG C; (7) cell washing liquid is added, 1200 turns/min, 10min low-temperature centrifugation, 2 times repeatedly, censorship after removal supernatant; (8) expression rate of HER2, P-HER2 albumen of flow cytomery SKOV3 cell.Above-mentioned experimental procedure in triplicate.
5, data analysis
Adopt the software of SPSS17.0 to carry out statistical analysis, represent with mean ± standard deviation (X ± s).Adopt one factor analysis of variance to compare measurement data, and compare employing LSD inspection between group between two, the comparison of rate adopts X 2 test, is that difference has statistical significance with P<0.05.
Three, result and conclusion
1, MTT experiment result
All occur obvious cell inhibitory effect effect after the CephaloziellinB of variable concentrations acts on people's adenocarcinoma ovaries SKOV3 cell 24,48 and 72h, show as OD value (absorbance A570) and reduce, suppression ratio raises; Result between variable concentrations group is variant, and difference has statistical significance (P<0.05), also significant difference (P<0.05) is had different action time, show that the propagation of CephaloziellinB to ovarian cancer SKOV3 cell is inhibited, and its suppression ratio is concentration and time dependence.Learnt by experimental result, when concentration is 20 μm of ol/L, effect is more satisfactory.The results are shown in Table 1.
2, PI staining flow cytometry (FCM) detects the result of cell cycle
The CephaloziellinB choosing variable concentrations (10,20,30 μm of ol/L) acts on SKOV348h, and experimental group comparatively matched group compares G
0/ G
1cell quantity in increasing trend, and S phase and G
2/ M cell quantity is minimizing trend, and when being 20 μm of ol/L with CephaloziellinB concentration, effect is the most obvious.Compared with matched group, each experimental group has statistical significance (P<0.05).G after each experimental group effect 48h
0/ G
1cell quantity be respectively 10 μm of ol/L and (60.707 ± 2.382), 20 μm of ol/L groups (69.611 ± 2.366), 30 μm of ol/L (61.099 ± 1.577), the analysis showed that 30 μm of ol/L groups are compared with 10 μm of ol/L, no significant difference (P=0.809), comparing difference between all the other each concentration groups all has statistical significance (P<0.05).After each experimental group effect 48h, the cell quantity of S phase is respectively l0 μm of ol/L group (24.254 ± 3.244), 20 μm of ol/L groups (19.468 ± 0.580), 30 μm of ol/L (17.743 ± 1.311), wherein 30 μm of ol/L groups are compared with 20 μm of ol/L groups, no significant difference (P=0.305), all the other each concentration groups compare difference statistical significance (P<0.05).G after each experimental group effect 48h
2the cell quantity of/M phase is respectively l0 μm of ol/L group (13.276 ± 0.658), 20 μm of ol/L groups (10.624 ± 0.483), 30 μm of ol/L (21.147 ± 2.865), and comparing difference between remaining each concentration group has statistical significance (P<0.05).It can thus be appreciated that the effect that the CephaloziellinB of 20 μm of ol/L acts on SKOV348h is best, by cell cycle arrest in G
0/ G
1phase, make it can not enter the S phase.The results are shown in Table 2 (note: compared with matched group, equal P<0.05; * G
0/ G
1during the phase, 30 μm of ol/L groups are compared with 10 μm of ol/L groups, no significant difference (P=0.809), and during S phase, 30 μm of ol/L are compared with 20 μm of ol/L, no significant difference (P=0.305); All the other G
0/ G
1, S, G
2compare between each phase group of/M, equal P<0.05.)。
3, PI staining flow cytometry (FCM) detects the expression of HER2, P-HER2
The expression of flow cytomery SKOV3 cell HER2 and P-HER2 on its surface before and after application CephaloziellinB, learn that the CephaloziellinB action effect of 20 μm of ol/L is ideal from above-mentioned experimental result, therefore this step adopts CephaloziellinB (20 μm of ol/L) to act on SKOV3 cell 48h, the change of HER2 protein expression not obvious (P=0.13) before and after result display application CephaloziellinB, and the expression of P-HER2 albumen has obvious difference (P<0.05).The results are shown in Table 3 (note: compared with matched group, * P=0.13,
#p<0.05).
Conclusion, CephaloziellinB can suppress the proliferation activity of ovarian cancer SKOV3 cell, and the result of this experiment MTT shows, and it affects the distribution of cell cycle, and tumor cell is arrested in G
0/ G
1phase, simultaneously by being combined with the tyrosine kinases phosphorylate ATP-binding site of EGFR/HER2 receptor, suppress its phosphorylation, and then the expression of lowering HER2, P-HER2 albumen carrys out cell death inducing.
Table 1 variable concentrations, the CephaloziellinB of different action time are on the impact of SKOV3 cell
The CephaloziellinB of table 2 variable concentrations acts on the impact of SKOV3 cell 48h cell cycle distribution
The CephaloziellinB of table 320 μm ol/L acts on the expression of HER2, P-HER2 after SKOV3 cell 48h
Claims (2)
- The application of 1.CephaloziellinB in preparation treatment ovarian cancer, described CephaloziellinB chemical structural formula is as follows,
- 2. the application of CephaloziellinB according to claim 1 in preparation treatment ovarian cancer, is characterized in that: described ovarian cancer is SKOV3.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105481874A (en) * | 2015-12-22 | 2016-04-13 | 陈杰 | Novel diterpene compound for treating ovarian cancer |
| CN105602898A (en) * | 2016-03-31 | 2016-05-25 | 谷超 | Animal origin-free culture medium capable of efficiently amplifying human lymphocytes |
| CN105748726A (en) * | 2016-04-29 | 2016-07-13 | 陈芝维 | Chinese herb compound for treating ovarian cancer and preparation method thereof |
| CN106146291A (en) * | 2016-07-04 | 2016-11-23 | 郑飞珍 | A kind of new sesquiterpene carboxylic acid compound and preparation method thereof and medical usage |
-
2015
- 2015-09-13 CN CN201510578917.4A patent/CN105055395A/en not_active Withdrawn
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105481874A (en) * | 2015-12-22 | 2016-04-13 | 陈杰 | Novel diterpene compound for treating ovarian cancer |
| CN105602898A (en) * | 2016-03-31 | 2016-05-25 | 谷超 | Animal origin-free culture medium capable of efficiently amplifying human lymphocytes |
| CN105748726A (en) * | 2016-04-29 | 2016-07-13 | 陈芝维 | Chinese herb compound for treating ovarian cancer and preparation method thereof |
| CN106146291A (en) * | 2016-07-04 | 2016-11-23 | 郑飞珍 | A kind of new sesquiterpene carboxylic acid compound and preparation method thereof and medical usage |
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Application publication date: 20151118 |