CN106390098A - Composition comprising oroxylin and used for treating leukemia and application of composition - Google Patents

Composition comprising oroxylin and used for treating leukemia and application of composition Download PDF

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Publication number
CN106390098A
CN106390098A CN201610994622.XA CN201610994622A CN106390098A CN 106390098 A CN106390098 A CN 106390098A CN 201610994622 A CN201610994622 A CN 201610994622A CN 106390098 A CN106390098 A CN 106390098A
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oroxylin
cell
tnf
composition
leukemia
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郭青龙
魏立彬
惠慧
卢娜
赵丽
周煜新
张晓博
赵凯
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China Pharmaceutical University
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China Pharmaceutical University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline 
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/191—Tumor necrosis factors [TNF], e.g. lymphotoxin [LT], i.e. TNF-beta

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Epidemiology (AREA)
  • Medicinal Chemistry (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Zoology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Immunology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

The invention belongs to the field of antineoplastic medicaments and discloses a composition comprising oroxylin and used for treating leukemia and application of the composition. Active ingredients of the medicine composition contain the oroxylin and tumor necrosis factors. The medicine composition has an excellent therapeutic effect on the leukemia, can be used to prepare anti-leukemia medicines and is high in clinical value.

Description

One kind is used for treating leukemic composition and its application containing oroxylin
Technical field
The invention belongs to field of antineoplastic medicaments, be related to a kind of containing oroxylin be used for treating leukemic composition and its Application.
Background technology
Oroxylin (Oroxylin A) is one of active component of the root of large-flowered skullcap, has obvious antitumor action, is one There is the natural antitumor medicine of broad prospect of application.Many documents are to the antitumor action of oroxylin and apoptosis-induced at present Carry out systematic research with Cycle Arrest, found that oroxylin has to the human liver cancer cell HepG2 of in vitro culture and significantly give birth to Long inhibitory action, and to ICR mouse experiment transplantable tumor S180Also there is significant growth inhibition effect, inhibiting rate reaches as high as More than 40%, act on low differentiation BGC823 cell line BGC-823 and lead to its Cycle Arrest in the G2/M phase, point out oroxylin With inducing apoptosis of tumour cell, and can be directed to Cell cycle checkpoint modulate tumor cell growth.But currently without The research report that oroxylin affects on leukaemia.
Content of the invention
It is an object of the invention to provide a kind of anti-leukocythemia composition containing oroxylin.
It is a further object to provide application in preparation treatment leukemia medicament for the above-mentioned composition.
The treatment of ATRA (ATRA) is invalid to the non-acute progranulocyte leukemia such as U937 and HL-60, TNF α can promote U937 and HL-60 to break up, but has obvious side effect to suppress its differentiation effect in atomization, and oroxylin is combined TNF α can the reaction to TNF α for the sensitizing cells.
The purpose of the present invention is realized by following technical proposal：
A kind of pharmaceutical composition, the active component of this pharmaceutical composition comprises oroxylin and TNF.
The active component of described pharmaceutical composition contains the component of following concentration ratio：
10～50 μM of oroxylin：TNF 10～20ng/mL.
Described pharmaceutical composition treats the application in leukemic medicine in preparation.
Described leukaemia is acute myeloid leukemia.
Beneficial effects of the present invention：
The present invention has been experimentally confirmed oroxylin and can strengthen TNF α suppression human leukemia cell line with TNF α combination The vigor of NB4, cell line U937-MDR and cell line HL-60-resistant, also can strengthen TNF α suppression people AML primary thin The vigor of born of the same parents, the life cycle of prolonged human AML primary cell Mice Inoculated, can be used for preparation and treats leukemic medicine, have relatively Good potential applicability in clinical practice.
Brief description
Fig. 1：Oroxylin strengthens the suppression to AML cell viability for the TNF α.
Fig. 2：Oroxylin strengthens the suppression to AML cell viability for the TNF α.
Fig. 3：TNF α is combined the life cycle of oroxylin prolonged human AML primary cell Mice Inoculated.
Specific embodiment：
The invention will be further elaborated by the following examples.
Embodiment 1
Flavones anti-tumor compounds are mainly combined by this research with antineoplastic TNF (Tumor Necrosis Factor) alpha, are used for Treatment acute myeloid leukemia (AML).Found by vivo and in vitro, oroxylin can be with TNF α synergy.
1st, experiment material
1.1 reagent
(1) oroxylin (oroxylin A) is purchased from Jiangsu Province's tumour and occurs and intervention laboratory (middle traditional Chinese medicines University of Science and Technology Learn), using front use dimethyl sulfoxide (DMSO), drug powder is formulated as the mother liquor of the concentration of 0.5M, uses cell culture fluid before use It is made into desired concn.ATRA (ATRA) is purchased from Sigma Co., USA, and being dissolved in DMSO and being configured to concentration is 20mM's Mother liquor.
(2) recombinant human tumor necrosis factor α (TNF α) is purchased from Prospec-Tany TechnoGene limited company. Using the front mother liquor that first with sterilized water, powder is configured to 1.0mg/ml concentration, then it is diluted to 1 μ g/ml's with 0.1%BSA Storing solution, -20 DEG C of packing preserve.It is made into desired concn with cell culture fluid before use.
(3) cell culture reagent
1. nutrient solution：RPMI-1640 culture medium, purchased from GIBCO company of the U.S..RPMI-1640 powder 10.39g is taken to be dissolved in In 1000ml sterilizing tri-distilled water, and add 2.0g NaHCO3, with 1M hydrochloric acid tune pH value to 7.0, cylindric style filter is crossed and is filtered Bacterium, packing, 4 DEG C of Refrigerator stores.Using the front addition penicillin of 100U/ml and the streptomysin of 100U/ml.
2. hyclone：U.S.'s GIBCO Products.Inactivate 30min through 56 DEG C of water-baths, dispense and be stored in -20 DEG C low In temperature refrigerator.
3. PBS：Weigh NaCl 8.0g, KCl 0.20g, Na2HPO4·H2O 1.56g、KH2PO4 2.0g, molten In 1000ml tri-distilled water, autoclaving, 4 DEG C of Refrigerator stores.
(4) lymphocyte separation medium, the brilliant U.S. in Nanjing Products.
(5) MTT reservoir (5mg/ml is dissolved in PBS)：Sigma Co., USA's product.MTT powder 1.0g is taken to be dissolved in 20ml PBS In solution, lucifuge ultrasonic dissolution.
(6) cell differentiation detection related reagent
People anti-mouse CD11b-PE, anti-CD14-FITC antibody is purchased from Miltenyi company.
Bovine serum albumin(BSA) (Bovine Serum Albumin, BSA) is purchased from Roche company of Switzerland.
(7) ATRA (ATRA) is purchased from Sigma Co., USA, is dissolved in DMSO and is configured to the mother that concentration is 20mM Liquid.
(8) oroxylin (oroxylin A) is purchased from Jiangsu Province's tumour and occurs and intervention laboratory (middle traditional Chinese medicines University of Science and Technology Learn), make intravenous injection.
(9) recombinant human tumor necrosis factor α (TNF α) (500,000IU/ml) is purchased from the limited public affairs of Shanghai dimension section biological medicine Department.Mouse mainline (3,000IU/kg).
1.2 laboratory apparatus
(1) YJ-875 type Medical purification workbench：Suzhou Decontamination Equipment Plant produces.
(2) 3111 type water-jacket typ CO2 incubators：U.S.'s Thermo electron Products.
(3) electronic balance：Beijing Sai Duolisi instrument system Co., Ltd product.
(4) ELX800 enzyme-linked immunosorbent assay instrument：U.S.'s Bio-tek Products.
(5) QIUJING blood cell counting plate：Chinese Shanghai refinement biochemical reagents Instrument Ltd. product.
(5) Olympus camera：Japanese Olympus Products.
(6) LD4-2 generic centrifuge：Beijing Medical Centrifugal Machine Factory's product.
(7) the desk-top high-speed refrigerated centrifuge of 5417R type：German Eppendorf Products.
(8) THZ-312 type Desk type constant-temperatureoscillator oscillator：Upper Nereid grand testing equipment Co., Ltd product.
1.3 cell line
Human leukemia cell line NB4 is purchased from cell institute of the Shanghai Chinese Academy of Sciences, cell line U937-MDR and HL-60- Resistant is by Alberta. and the Robert E.Gallagher professor laboratory of Einstein medical college builds, by Guangzhou Ji Niou bio tech ltd buys.People's AML primary cell is bought in Jiangsu Prov. People's Hospital hematology.All cells are equal RPMI1640 nutrient solution culture with penicillin containing 100U/ml, 100mg/ml streptomysin and 10% hyclone.
NB4 is acute promyelocytic leukemia (APL) cell line, and U937-MDR is U937 MDR strain, HL-60- Resisitant is the ATRA ATRA persister of HL-60.Differentiating inducer ATRA (ATRA) has to NB4 Effect, as positive control；U937 and HL-60 is the acute myeloid leukemia (AML) of non-acute promyelocytic leukemia, all Cannot respond to the treatment of ATRA (ATRA).
1.4 animal used as test
Strain：NOD/SCID mouse；
Week old：6-8 week；
Body weight：18-22g；
Rearing conditions：Pellet；SPF air-conditioned room, temperature 18-24 DEG C, relative humidity 70%.
Purchased from Chinese Academy of Sciences's Shanghai Experimental Animal Center.
2nd, experimental technique
2.1 lymphocyte separation mediums separate human leukemia cell
In human peripheral, the density of various cells is different, and HRBC's density is 1.093, and granulocyte is 1.092, and lymph is thin Born of the same parents are 1.074 ± 0.001.The principle of the density-gradient centrifugation method mainly difference according to all kinds of haemocyte proportions, using than dense medium Cell separation liquid between certain two class cell, to centrifugal blood, makes the haemocyte of certain weight proportion be distributed by corresponding density gradient In different free bands, thus reaching detached purpose.2ml lymphocyte separation medium will be added in aseptic 10ml centrifuge tube. After venous blood in 2ml anticoagulant heparin pipe (or sodium citrate) and equivalent serum free medium are fully mixed, use pipettor edge Tube wall is slowly added dropwise on laminated fluid level, notes keeping clearly interface.2000rpm is centrifuged 20min.It is divided into three in pipe after centrifugation Layer has a white cloud and mist narrow band based on mononuclearcell in upper, middle level interface.Mononuclearcell includes lymphocyte And monocyte.It is inserted into cloud and mist layer with capillary or suction nozzle, draw mononuclearcell.It is subsequently placed in another centrifuge tube.Add 5 The free serum culture base fluid washing of times volume, turns upside down and mixes for several times, 1500rpm is centrifuged 15min.Abandon supernatant, repeat and wash Wash cell 1 time.After final centrifugation, abandon supernatant, as far as possible supernatant exhausts.1ml is added to contain 10% calf serum RPMI1640, re-suspended cell.Use immediately after cell separation is good.Centrifugation schematic diagram is as follows：
2.2MTT experiment
MTT can be reduced to hepatic crystallization first, the depth of its lysate color by the mitochondrial dehydrogenase in living cells The shallow vigor size with cell is proportional.The cell being in exponential phase needed for experiment is inoculated in 96 holes by certain concentration In ELISA Plate.Cell implantation concentrations are typically 0.5～2 × 104Individual/hole, every in the hole volume is 50 μ l.And add 50 μ l final concentrations 20 μM of oroxylin pretreatment 24h.The TNF α being subsequently added into 100 μ l difference final concentration (10,20ng/mL) continues to be placed in incubator Inside cultivate to 96h, every hole adds 20 μ l MTT solution.After continuing incubation 4h, the supernatant in every hole is exhausted.Every hole adds 100 μ l DMSO, being put in vibration 5min on microoscillator makes crystallization dissolve.To be crystallized be completely dissolved after be put on ELIASA inspection Survey, Detection wavelength is 570nm.The growth inhibition to cell for the medicine is calculated by equation below after light absorption value after detection is arranged Rate.
2.3 streaming surface antigen detections
Break up with cell, surface of cell membrane proteantigen also accordingly changes, these eggs relevant with differentiation It is referred to as differentiation antigen in vain.Can more accurately judge that whether cell breaks up by detecting cell surface differentiation antigen. Wherein, CD11b is the ripe mark antigen of myeloid differentiation.TNF α combination oroxylin or ATRA act on human cell line and people Collect cell after AML primary cell incubation 96h and count, each group cell is adjusted to same density (5 × 105Individual/ml), with containing The nonspecific binding site on the PBS closing cell surface of 0.5%BSA, resuspended after centrifugation, add antibody, so that PE is marked The CD14 antibody of the CD11b and FITC mark of note is combined with the T cell differentiation antigen of cell surface to be measured, then uses immunofluorescent flow cell Metering art (FCM) counts fluorescently-labeled positive cell, compares with the cell through the process of same processing mode not adding antibody, And make Isotype control.
2.3 people's AML primary cell NOD/SCID Establishment of mouse model methods
(1) take patient's AML Fresh blood sample, isolate mononuclearcell, free serum culture with lymphocyte separation medium Base RPMI 1640 is resuspended stand-by.
(2) animal irradiation.NOD/SCID mouse is carried out x-ray irradiation, irradiation is 2.4Gy.
(3) inoculated in 24 hours after irradiation, resuspended after primary cell is centrifuged is 1 × 10 for final densities7Individual/ Cell, with empty needle suction, is mixed, with 0.1ml (1 × 10 before aspirating every time by ml6Individual) it is inoculated in NOD/SCID mouse tail vein.
(4) test sets blank group, negative control group, single medicine group and combination group.Blank group, negative control group is to physiology salt Water, the TNF α of oroxylin or 3,000IU/kg that single medicine group gives 80mg/kg (is changed with mouse by people's Clinical practice dosage Calculate), oroxylin lumbar injection is administered every other day, and TNF α tail vein injection is administered, weekly the administration of Wednesday to Saturday, to common administration 30 My god.
(5) during being administered, observe the survival condition of each group mouse.Record dead mouse situation.
(6) arrange result, provide report.
3rd, experimental result
(1) the TNF α combination impact to AML cell line cell viability for the oroxylin
The TNF α (10,20ng/mL) of variable concentrations is measured by MTT and 20 μM of oroxylins are each alone or both are combined After 4 days, the change of NB4, U937-MDR and HL-60-resistant cell viability.Experimental result shows (Figure 1A), uses with independent Medicine group is compared, and TNF α is combined the vigor that oroxylin significantly suppresses three kinds of cells.Equally, in people's AML primary cell, TNF α Combination oroxylin also can significantly inhibit cell viability (Figure 1B).
(2) TNF α is combined the impact that oroxylin breaks up associated surface antigens to AML cell line
As shown in Fig. 2 compared with independent medication group, after TNF α combination oroxylin administration, NB4, U937-MDR and HL- Two kinds of surface antigens of 60-resistant cell surface CD11b and CD14 all substantially increase, and difference is all statistically significant.Say Bright, TNF α combination oroxylin can induce these three leukaemias to ripe direction change, and has the differentiation of monokaryon system Feature.Meanwhile, after the administration of TNF α combination oroxylin, people's two kinds of surface antigens of primary AML cell surface CD11b and CD14 are also sent out Raw substantially increase, differentiation capability significantly improves.
(3) TNF α is combined the impact that oroxylin inoculates NOD/SCID mouse model in vivo to people's AML cell
As shown in figure 3, compared with negative control group, it is primary that TNF α combination oroxylin can significantly extend inoculation people AML The life cycle of the NOD/SCID mouse of cell.

Claims (4)

1. a kind of pharmaceutical composition is it is characterised in that the active component of this pharmaceutical composition comprises oroxylin and tumor necrosis factor Son.
2. pharmaceutical composition according to claim 1 it is characterised in that the active component of this pharmaceutical composition contain following The component of concentration relationship：
10～50 μM of oroxylin：TNF 10～20ng/mL.
3. the pharmaceutical composition described in claim 1 or 2 treats the application in leukemic medicine in preparation.
4. application according to claim 3 is it is characterised in that described leukaemia is acute myeloid leukemia.
CN201610994622.XA 2016-11-11 2016-11-11 Composition comprising oroxylin and used for treating leukemia and application of composition Pending CN106390098A (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112754991A (en) * 2020-12-03 2021-05-07 中国药科大学 Oroxylin injection and application thereof in preparation of liver cancer drugs

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101244055A (en) * 2008-03-03 2008-08-20 中国药科大学 Application of oroxylin in pharmacy

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101244055A (en) * 2008-03-03 2008-08-20 中国药科大学 Application of oroxylin in pharmacy

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
XU ET AL.: "Oroxylin a Heightens Tumor Necrosis Factor a-Induced Differentiation Effects on Acute Myeloid Leukemia Cells Via Modulation on AKT and NF-Kb Pathways Involving RXRa", 《BLOOD》 *
王玉等: "千层纸素诱导人慢性粒细胞白血病K562 的凋亡作用", 《中国药科大学学报》 *
陈妍等: "千层纸素通过调节核受体异源二聚体ppar/rxr从而诱导白血病细胞发生分化和G0/G1期周期阻滞", 《中国实验血液学杂志》 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112754991A (en) * 2020-12-03 2021-05-07 中国药科大学 Oroxylin injection and application thereof in preparation of liver cancer drugs
CN112754991B (en) * 2020-12-03 2022-05-17 南京芩领医药科技有限公司 Oroxylin injection and application thereof in preparation of liver cancer drugs

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Application publication date: 20170215