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CN107049998A - Application of the lycopene cis-isomer in treatment of ovarian cancer medicine is prepared - Google Patents

Application of the lycopene cis-isomer in treatment of ovarian cancer medicine is prepared Download PDF

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Publication number
CN107049998A
CN107049998A CN201610846435.7A CN201610846435A CN107049998A CN 107049998 A CN107049998 A CN 107049998A CN 201610846435 A CN201610846435 A CN 201610846435A CN 107049998 A CN107049998 A CN 107049998A
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lyp
lycopene
cis
apoptosis
group
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CN201610846435.7A
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Inventor
李�荣
周玉生
许时丽
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Second Hospital University Of South China
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Second Hospital University Of South China
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    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00Medicinal preparations containing organic active ingredients
    • A61K31/01Hydrocarbons

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  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)

Abstract

The Carbazole alkaloids of oophoroma HO 8910 are bred the invention discloses All-cislycopene (Lycopene, Lyp) isomers and apoptosis-induced effect, belong to pharmaceutical technology field.Research shows that the incidence of Lyp concentration and prostate cancer, human primary gastrointestinal cancers, breast cancer, carcinoma of urinary bladder etc. is negatively correlated in blood, but whether plays the role of inhibitory action and cis and trans isomers to oophoroma whether variant there is not been reported.Thus, inventor proposes hypothesis first:By alltrans Lyp can isomerization obtain cis Lyp, and the two is to the Carbazole alkaloids of oophoroma HO 8910 propagation and apoptosis-induced having differences property of effect.In the present invention, inventor confirms above-mentioned hypothesis with cell experiment, and the effect that cis Lyp suppresses the cells of HO 8910 in vitro is more stronger than alltrans Lyp, and the two concentration is in the μ gmL of 10 μ~20‑1When, inhibiting rate and apoptosis rate have significant difference.All-cislycopene can as treatment of ovarian cancer drug candidate.

Description

Application of the lycopene cis-isomer in treatment of ovarian cancer medicine is prepared
Technical field
The present invention relates to a kind of new application of isomers of lycopene, and in particular to prepared by lycopene cis-isomer Application in oophoroma HO-8910 cells propagation and Apoptosis medicine, belongs to pharmaceutical technology field.
Background technology
Lycopene (Lycopene, Lyp) has quenching activity oxygen, eliminates human free radical, prevents heart disease, slows down dynamic The physiological function such as pulse atherosclerosis, prevention kinds cancer, anti-aging, protection skin.Epidemiological study shows, Lyp in blood The incidence of concentration and prostate cancer, cancer of the esophagus, cancer of pancreas, human primary gastrointestinal cancers, breast cancer, cutaneum carcinoma, carcinoma of urinary bladder etc. is negatively correlated.Perhaps Many In vitro cell experiments all confirm this point, and Lyp has suppression growth to esophageal carcinoma cell line (Eca9706), before people Row gland cancer PC-3 cells and breast cancer MDA-MB-231 cells have Inhibit proliferaton and apoptosis-induced effect, thin to cervical cancer Hela cells Born of the same parents and mice model of forestomach tissue canceration also have certain inhibitory action.But whether Lyp has inhibitory action to the oophoroma for having high incidence See that there is not been reported before periodical in the present inventor's research paper.
Lyp can reduce the activity of breast cancer cell mitogen, suppress cell cycle arrest in the G1 phases to S phases, Lyp can increase Strong GJIC (GJIC) function, promotes the interaction between phagocyte, lymphocyte, is lived by secretory cell Change factors activated cell, be eventually exhibited as the promotion to phagocytic activity and lymphocyte transformation, strengthen immunologic function and then induction Apoptosis of tumor cells.A research report report that NATUER in 2012 is delivered:Breast cancer genetic analysis show its procarcinogen because It is similar to oophoroma.
The content of the invention
It is an object of the invention to provide application of the lycopene cis-isomer in treatment of ovarian cancer medicine is prepared.
Lycopene structure formula
Lycopene, molecular formula C40H56, relative molecular mass 536.85 is a kind of important carotenoid in meals, mainly It is present in tomato products, watermelon, pomegranate, autumn olive and purple grape fruit.The lycopene of natural origin is main with complete Trans forms are present, and are most stable of structures.In human plasma, lycopene content is 0.2-1.0 μm of ol/L, mainly with suitable Formula configuration is present, and anti-configuration only accounts for 41%.Foreign study proves that All-cislycopene is easier to be absorbed by the body, it may have Higher bioactivity.
Both at home and abroad in lycopene and the relation document of cancer, difference between the effects research to cis and trans isomers there is not yet Report.Thus, inventor proposes hypothesis first:By trans lycopene can the obtained All-cislycopene of isomerization, and the two To human ovarian cancer cell line HO-8910 Inhibit proliferaton and apoptosis-induced having differences property of effect.In the present invention, demonstrate,proved with cell experiment Real above-mentioned hypothesis, Preliminary Results prompting suppresses the effect of HO-8910 cells than complete in vitro containing Lyp cis at high proportion Trans Lyp is more stronger, and the two concentration is in 10 μ gmL-1With 20 μ gmL-1When, inhibiting rate and apoptosis rate relatively have significantly Sex differernce.
All-cislycopene by all-trans lycopene isomerization using being prepared, with reference to Pierre Lambelet etc. side Method carries out cisization processing:Weigh the lycopene powder being purchased from appropriate, dissolved, be made with the ethyl acetate solution through membrane filtration 1mg·mL-1Solution, 100 DEG C of water-baths of lucifuge backflow 5h place 12h under the conditions of 4 DEG C, filter, remove crystallization (I medicines, entirely Trans lycopene), filtrate decompression is evaporated to dryness, and obtains red acicular crystal (II medicines, All-cislycopene powder).
Lycopene cis-isomer is not yet used to prepare medicine at present, and the formulation of health products is capsule and tablet.
Embodiment
1 materials and methods
1.1 experiment material
Lycopene powder (purity 90%) (Huabei Pharmaceutic Co., Ltd)
HO-8910 cell lines (Xiangya Hospital, Central-South China Univ.'s cell centre)
Tetrazolium (Sigma companies)
0.4% trypan blue (Gibco companies)
RPM-1640 (Gibco companies)
Newborn calf serum (Hangzhou Chinese holly)
Docetaxel injection (DOC, permanent auspicious medicine)
Dimethyl sulfoxide (DMSO) (DMSO)
Acetonitrile, tetrahydrofuran, ethyl acetate (Chemical Reagent Co., Ltd., Sinopharm Group)
The synthesis of 1.2 All-cislycopenes is with separating
Cisization processing is carried out with reference to Pierre Lambelet etc. method:Weigh the lycopene purchased from North China drugmaker Appropriate powder, is dissolved with the ethyl acetate solution through membrane filtration, 1mgmL is made-1Solution, 100 DEG C of water-baths of lucifuge backflow 5h, 12h is placed under the conditions of 4 DEG C, is filtered, crystallization (I medicines) is removed, filtrate decompression is evaporated to dryness, and obtains red acicular crystal (II Number medicine).
The preparation of lycopene oil solution and uv scan:By document[5]Method, weighs I, II medicine and tomato red respectively Plain each 0.0200g of powder is put in 100mL measuring bottles, and each bottle first adds DMSO0.5ml, adds sunflower oil and is diluted to scale, shakes It is even, storing solution is made.Precision measures storing solution 1mL plus sunflower oil is settled in 100mL measuring bottles, shakes up, in 200~600nm Place's scanning ultra-violet absorption spectrum.
I, II medicine oil solution are separated by HPLC:By literature method, I, II medicine oil solution are passed through into HPLC separation detections Purity, chromatographic condition is chromatographic column:COSMOSIL Cholester posts (4.6mm × 250mm, 5um), mobile phase is tetrahydrofuran : acetonitrile=15: 85 (v/v), sample size 20uL, flow velocity 1mlmin-1, 15 DEG C of column temperature, Detection wavelength 472nm.With lycopene Powder and I medicines are as reference substance, and 0.1mgmL containing lycopene is made as test sample in II medicines-1Reference substance and confession Test sample solution.
1.3HO-8910 the culture and packet of cell
37 DEG C, 5%CO are based on the RPMI1640 cultures containing 10% calf serum2Cultivated under saturated humidity.Cell in culture medium Adherent growth, growth period cell of taking the logarithm is used to test.Experiment sets 9 group (1) blank control groups:Only contain nutrient solution;(2) solvent Control group:Nutrient solution containing 0.5%DMSO;(3) II medicines low dose group:The μ gmL of All-cislycopene content about 5-1;(4)II Number medicine middle dose group:The μ gmL of All-cislycopene content about 10-1;(5) II medicines high dose group:All-cislycopene content is about 20μg·mL-1;(6) I medicines low dose group:The μ gmL of trans lycopene content about 5-1;(7) I medicines middle dose group:It is trans The μ gmL of lycopene content about 10-1;(8) I medicines high dose group:The μ gmL of trans lycopene content about 20-1;(9) it is positive Medicine control group:Final concentration of 0.2 μ gmL-1DOC solution.
The measure of 1.4HO-8910 cell growth curves
Collect exponential phase HO-8910 cells 5 × 105/ L is inoculated in 24 well culture plates, per hole 1ml, is then distinguished per hole 1.3 lower each group solution are added, 37 DEG C, 5%CO is put2Cultivated in incubator, and make cell count in 24,48,72,96,120h. During counting, check cell survival rate more than 95% with 0.4% trypan blue.Every group of 3 multiple holes, take its average value to draw growth bent Line.
1.5MTT method
Growth period HO-8910 cell of taking the logarithm is made 1 × 105Individual/L single cell suspension, is inoculated in 96 well culture plates, treats thin 1.3 lower each group solution are separately added into after born of the same parents are adherent per hole, cumulative volume is, per hole 200ul, 4 multiple holes to be set per dose, in 48h Afterwards, 5mg/mlMTT20 μ l are added per hole to continue to be incubated 4h, discard nutrient solution, dimethyl sulfoxide (DMSO) (DMSO) 100 μ l are added per hole, Fully mix, ELIASA (EX-800) surveys absorbance (A) value at 570nm, and experiment is repeated 3 times.Calculate average A-value and suppression Rate:Inhibiting rate (inhibitory, IR)=[(solvent control group A values-experimental group A values)/solvent control group A values] × 100%.
1.6 flow cytometer PI methods detect that suitable, anteiso- structure lycopene sunflower oil solution is thin to human ovarian cancer HO-8910 Born of the same parents' apoptosis rate difference growth period HO-8910 cell of taking the logarithm is made 1 × 106Individual/L single cell suspension, is inoculated in 6 well culture plates In, it is separately added into per hole after 1.3 lower each group solution, culture 48h, collects cell, PBS washings is made single cell suspension, used The 70% fixed 12h of 4 DEG C of ice ethanol, 30min, FACSort fluidic cells are contaminated with qiagen rnase enzyme and the dye liquor of propidium iodide Instrument carries out cycle analysis, is apoptotic cell less than G1 phase DNA contents cell.
1.7 statistical analysis
Data withRepresent, taken statistics analysis with SPSS11.0 softwares, two sample averages compare to be examined using t, with P < 0.05 There is significant for difference.
2 results
The Qualitive test of 2.1I, II medicine:By the contrast of ultraviolet spectrogram, I medicines can be assert with lycopene powder For all-trans lycopene, the cis peak at 362nm reported in the literature is occurred in that in II medicines.
The purity testing of 2.2I, II medicine:By HPLC method separation detection purity, calculated using quantified by external standard method, with kind Lycopene powder is as reference substance, and it is 95.82% to try to achieve alltrans Lyp contents in I medicines;Using I medicines as reference substance, try to achieve Cis Lyp contents are 79.53% in II medicines.
2.3 is suitable, anteiso- structure lycopene sunflower oil solution is to the ratio of human ovarian cancer cell line HO-8910 growth inhibition curve Compared with
Blank control group is consistent with solvent control group cell growth, shows 0.5%DMSO on the growth of cell without influence;I medicines Substantially it is suppressed compared with blank control group with the cell growth of each dosage group of II medicines and positive drug control group;Each experiment Group is in set concentration range, and with the increase of dosage, its inhibitory action strengthens;Growth inhibition of two high dose groups to cell Effect is remarkably reinforced compared with positive drug control group;I medicines low dose group to the growth inhibition effect of cell and II medicine phases ratio without Notable difference, but middle and high dosage I medicines group is weak compared with II medicines group suppression cell growth effect.See Fig. 1.
2.4 is suitable, anteiso- structure lycopene sunflower oil solution compares human ovarian cancer cell line HO-8910 proliferation inhibition rate
Solvent control group no significant difference (P > 0.05) compared with the absorbance of blank control group, illustrates 0.5%DMSO to thin Intracellular growth is without influence.Each experimental group of lycopene and the positive drug control group difference compared with blank control group have pole conspicuousness (P < 0.01).II medicines with the middle and high dosage group of I medicines is corresponding respectively compares, as a result with significant difference (P < 0.05).It is two high Dosage group is with there were significant differences compared with positive drug control group (P < 0.05).The basic, normal, high dosage group of II medicines is thin to HO-8910 Born of the same parents' proliferation inhibition rate is respectively 17.25%, 24.13%, 62.03%;The basic, normal, high dosage group of I medicines increases to HO-8910 cells It is respectively 16.86%, 17.57%, 54.05% to grow inhibiting rate;Positive drug control group is to HO-8910 cell proliferation inhibition rates 44.5%.It is shown in Table 1.
The inhibitory action that table 1 is suitable, anteiso- structure lycopene grows to human ovarian cancer cell line HO-8910
Note:Compared with blank control group,aP < 0.01;Compared with I medicine middle dose groups,bP < 0.05;With I medicine high dose groups Compare,cP < 0.05;With the μ gmL of DOC 0.2-1Group compares,dP < 0.05.
2.5 is suitable, comparison of the anteiso- structure lycopene sunflower oil solution to human ovarian cancer cell line HO-8910 Apoptosis
Solvent control group no significant difference (P > 0.05) compared with the apoptosis rate of blank control group.Each reality of lycopene Testing group and the positive drug control group difference compared with blank control group has pole conspicuousness (P < 0.01).II medicines and I medicines are middle and high Dosage group is corresponded to respectively to be compared, as a result with significant difference (P < 0.05) (P < 0.05).High dose group apoptosis rate is high In positive drug control group, difference has conspicuousness (P < 0.05).The basic, normal, high dosage group apoptosis rate of II medicines is respectively 11.5%th, 29.9%, 39.7%;The basic, normal, high dosage group apoptosis rate of I medicines is respectively 11.8%, 23.1%, 32.4%; Positive drug control group apoptosis rate is 24.6%.See Fig. 2.PI detection apoptotic cell scatter diagrams are shown in Fig. 3.
Recent study find, Lyp molecules from anti-configuration be changed into cis-configuration when, lighter, fusing point reduction, absorb Peak shifts, and heat treatment can dramatically increase the cis-Lyp in tomato juice.This experiment isomate process is consistent with document.This is thin Born of the same parents' experimental result shows that the effect for suppressing HO-8910 cells in vitro containing cis Lyp at high proportion is more stronger than alltrans Lyp, and The two concentration is in 10 μ gmL-1With 20 μ gmL-1When, inhibiting rate and apoptosis rate relatively have significant difference.
Suitable, anteiso- structure lycopene sunflower oil solution is to human ovarian cancer cell line HO-8910 growth inhibition curve and Apoptosis Relatively see Figure of description.Fig. 1 is the growth curve chart that suitable, anteiso- structure lycopene suppresses HO-8910 cells, Fig. 2 be it is suitable, The figure that anteiso- structure lycopene influences on human ovarian cancer cell line HO-8910 apoptosis rate, packet situation is:1. blank control group, 2. Solvent control group, 3.II medicine low dose groups, 4.II medicine middle dose groups, 5.II medicine high doses, 6.I medicine low dose groups, 7.I medicine middle dose groups, 8.I medicine high dose groups, the μ gmL of 9.DOC 0.2-1Group.Com-parison and analysis situation is:With blank control Group compares,aP < 0.01;Compared with I medicine middle dose groups,bP < 0.05;With I medicines;High dose group compares,cP < 0.05;With DOC 0.2μg·mL-1Group compares,dP < 0.05.Fig. 3 is apoptotic cell scatter diagram, and packet situation is:1. blank control group, 2. Solvent control group, 3.II medicine low dose groups, 4.II medicine middle dose groups, 5.II medicine high doses, 6.I medicine low dose groups, 7.I medicine middle dose groups, 8.I medicine high dose groups, the μ gmL of 9.DOC 0.2-1Group.

Claims (2)

1. application of the lycopene cis-isomer in treatment of ovarian cancer medicine is prepared.
2. apply according to claim 1, lycopene cis-isomer or its pharmaceutically acceptable salt or contain them Middle any pharmaceutical composition is preparing the application for the treatment of oophoroma.
CN201610846435.7A 2016-09-20 2016-09-20 Application of the lycopene cis-isomer in treatment of ovarian cancer medicine is prepared Pending CN107049998A (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111603458A (en) * 2020-06-24 2020-09-01 南华大学附属第二医院 A composition for treating ovarian cancer

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1095589A (en) * 1992-11-30 1994-11-30 马克特辛姆化学工厂有限公司 Reduce the method and the pharmaceutical preparation of cytoactive
CN1612728A (en) * 2001-11-12 2005-05-04 利库德天然产品工业有限公司 Method and pharmaceutical preparations for reducing the activity of cells

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1095589A (en) * 1992-11-30 1994-11-30 马克特辛姆化学工厂有限公司 Reduce the method and the pharmaceutical preparation of cytoactive
CN1612728A (en) * 2001-11-12 2005-05-04 利库德天然产品工业有限公司 Method and pharmaceutical preparations for reducing the activity of cells

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
SCOLASTICI C等: "Lycopene activity against chemically induced DNA damage in Chinese hamster ovary cells", 《TOXICOL IN VITRO》 *
李荣等: "番茄红素异构体对人卵巢癌HO-8910细胞增殖和凋亡的影响效果初探", 《中南药学》 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111603458A (en) * 2020-06-24 2020-09-01 南华大学附属第二医院 A composition for treating ovarian cancer
CN111603458B (en) * 2020-06-24 2022-12-16 南华大学附属第二医院 A composition for treating ovarian cancer

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Application publication date: 20170818