CN102068448A - Application of icariside II in preparation of anti-melanoma medicament - Google Patents
Application of icariside II in preparation of anti-melanoma medicament Download PDFInfo
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- CN102068448A CN102068448A CN2009101990883A CN200910199088A CN102068448A CN 102068448 A CN102068448 A CN 102068448A CN 2009101990883 A CN2009101990883 A CN 2009101990883A CN 200910199088 A CN200910199088 A CN 200910199088A CN 102068448 A CN102068448 A CN 102068448A
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Abstract
The invention belongs to the field of medicament preparation, relates to new medicinal application of icariside II, and particularly relates to application of icariside II in preparation of an anti-melanoma medicament. The icariside II is adopted for intervention experiments of mouse melanoma cell strains and human melanoma cell strains; by observing the inhibiting condition of the icariside II on cell proliferation, the results show that the icariside II can remarkably inhibit the proliferation of B16, A375 and SK-MEL-2 cells in vitro, obviously promote apoptosis of the cells and increase P53 protein phosphorylation and caspace protein expression; and the icariside II can remarkably inhibit loaded B16 mouse subcutaneous transplant tumor volume. Experiment results prove that the icariside II can effectively inhibit the melanoma. The icariside II can further be used for preparing the anti-melanoma medicament by adding proper pharmaceutically-acceptable carriers.
Description
Technical field:
The invention belongs to the medication preparation field.The new medical use that relates to icariside I I is specifically related to the purposes of icariside I I in preparation melanoma medicine.
Background technology:
Melanoma is a kind of clinical kinds of tumor, often is primary in skin, mucosa.Clinical practice is observed and is shown that its grade malignancy is very high, and metastasis easily takes place.It is all insensitive to radiotherapy and chemotherapy, and survival rate was very low in 5 years.
The screening active component is the new direction of melanoma treatment from natural drug.
Icariside I I is present in the epimedium herb on a small quantity, and its chemical structural formula is as follows:
Icariside I I
The pharmacology activity research report of at present relevant excessive sheep flower bud element is less.Chinese patent CN200780034447.9 discloses " application of icariside I I in preparation skin whitening medicine ".Yet do not appear in the newspapers as yet in the purposes aspect the preparation melanoma medicine about icariside I I at present.
Summary of the invention
The object of the present invention is to provide the new pharmaceutical usage of icariside I I, be specifically related to the application of icariside I I in preparation melanoma medicine.
The present invention adopts icariside I I to carry out mouse melanin tumor cell strain (B16) and human melanoma cell strain (A375, SK-MEL-2) intervention experiment, observe icariside I I on cell proliferation and suppress situation, cell proliferation inhibition rate=(1-(dosing group OD meansigma methods-zeroing hole OD meansigma methods)/(blank group OD meansigma methods-zeroing hole OD meansigma methods)) * 100%.The ICS intervention time is 24h, and experiment repeats more than 3 times.The result shows that 1. the external propagation that can significantly suppress B16, A375 and SK-MEL-2 cell of icariside I I obviously promotes its apoptosis, increases P53 protein phosphorylation and caspace protein expression; 2. icariside I I can significantly suppress lotus B16 mouse subcutaneous transplanting tumor volume.
Experimental result confirms that icariside I I can effectively suppress melanoma.
Icariside I I of the present invention can be further the medicine of suitable pharmaceutically acceptable preparing carriers melanoma in addition.
Among the present invention, the experiment cell strain that is adopted is commercially available biomaterial.
Among the present invention, adopt routine test method known in the art to carry out above-mentioned mouse melanin tumor cell strain (B16) and human melanoma cell strain (A375, SK-MEL-2) intervention experiment.
The new pharmaceutical usage of icariside I I of the present invention is for the treatment melanoma provides a kind of new approach and means.
Description of drawings
Fig. 1 is: icariside I I (ICS) is to the influence of three kinds of melanoma cell strain cell proliferation,
Wherein, the ICS concentration dose is μ M, and detection method is the WST-8 method, and Figure 1A is the influence of each concentration ICS to mouse melanin tumor cell strain B16 cell proliferation inhibition rate; Figure 1B is the influence of each concentration ICS to human melanoma cell strain A375 cell proliferation inhibition rate; Fig. 1 C is the influence of each concentration ICS to the cell proliferation inhibition rate of human melanoma cell strain SK-MEL.
Fig. 2 be icariside I I (ICS) to the apoptotic influence of human melanoma cell strain A375, wherein, the ICS concentration dose is μ M, detection method is that the two method streamings of dying of Annexin V and PI detect, the ICS intervention time is 24h.
Fig. 3 is icariside I I (ICS) to melanoma cell strain A375P-P53 with to the influence of human melanoma cell strain A375 Caspace-3 protein expression, wherein, Fig. 3 A, ICS is to the influence of human melanoma cell strain A375P-P53 protein expression; Fig. 3 B ICS is to the influence of human melanoma cell strain A375 Caspace-3 protein expression; The ICS concentration dose is μ M, and detection method is Westernblot, and the ICS intervention time is 24h.
Fig. 4 is the influence of icariside I I (ICS) to lotus B16 melanoma mouse tumor volume,
Wherein, DMSO is the contrast of lotus tumor, and ICSM is an icariside I I 50mg/kg group, and ICSH is an icariside I I 100mg/kg group, and D is a natural law after kind of the tumor; Administering mode is a lumbar injection, and administration time is that the measurement of tumor time is 3 times weekly 3 times weekly.
The specific embodiment
The experiment in vitro of embodiment 1 icariside I I melanoma cell strain
Materials and methods
1, cell strain: mouse melanin tumor cell strain (B16), available from Chinese Academy of Sciences's Shanghai cell bank.Human melanoma cell strain (A375 and SK-MEL-2) is available from ATCC.Above-mentioned cell strain is 5%CO with the DMEM culture medium that contains 10% hyclone at 37 ℃, volume fraction
2, complete conventional cultivation the under the saturated humidity condition, 48h changes culture medium, the cell growth reaches full when closing state, with 0.25% trypsin+0.02%EDTA had digestive transfer culture, 2～3d goes down to posterity 1 time, tests and selects the exponential phase cell for use.
2, the detection of cell proliferation:
Adjust B16, A375 respectively and SK-MEL-2 cell strain concentration of cell suspension is 1 * 10
5/ mL adds in 96 well culture plates, and every hole 100 μ L put 37 ℃ of 5%CO
2Incubator is cultivated 24h.Absorb old culture medium, add respectively and contain the serum-free DMEM culture medium (drug level is 100 μ M, 50 μ M, 25 μ M, 5 μ M) of icariside I I, and establish blank group and zeroing group, all establish 3 multiple holes, continue to cultivate 24h for every group.Add 10 μ LWST-8 solution, cultivate 2h.Detect the light absorption value in each hole at the 490nm place.Cell proliferation inhibition rate=(1-(dosing group OD meansigma methods-zeroing hole OD meansigma methods)/(blank group OD meansigma methods-zeroing hole OD meansigma methods)) * 100%.Experiment repeats more than 3 times.
3, apoptotic detection
Adjusting the A375 concentration of cell suspension is 1 * 10
6/ mL, with cell inoculation in 6 orifice plates, every hole 1ml.After treating that the cell growth reaches the fusion state, former culture medium is abandoned in suction, add respectively and contain icariside I I (drug level is 100 μ M, 50 μ M, 25 μ M, 5 μ M) serum-free DMEM culture medium, blank group adds the culture medium that contains 0.1%DMSO, behind the drug effect 24h, cell conditioned medium is drawn in the centrifuge tube, and cell moves in the centrifuge tube identical with cell conditioned medium after trypsinization, 4 ℃ of centrifugal 5min of 1000rpm, abandon supernatant, wash 1 time, abandon supernatant with PBS, wash 1 time with Binding buffer, and resuspended with 195 μ L Binding buffer, the room temperature lucifuge was hatched 10 minutes behind the 5 μ LAnnexin V-FITC mixings, washed 1 time with Binding buffer, and resuspended with 190 μ L Binding buffer, add behind the 10 μ L PI mixings machine testing on the flow cytometer.
4, the detection of P-P53, Caspace3 protein expression
Adjusting the A375 concentration of cell suspension is 1 * 10
6/ mL, with cell inoculation in 6 orifice plates, every hole 1ml.After treating that the cell growth reaches the fusion state, former culture medium is abandoned in suction, add respectively and contain icariside I I (drug level is 5 μ M, 10 μ M, 25 μ M, 50 μ M) serum-free DMEM culture medium, blank group adds the culture medium that contains 0.1%DMSO, collecting cell behind the drug effect 24h, extracting albumen, quantitatively back row SDS-PAGE electrophoretic separation, treat to be transferred to nitrocellulose filter after electrophoresis finishes, sealing at room temperature adds P-P53 or Caspace3 one is anti-, and 4 ℃ are spent the night, it is anti-to add HRP labelling two, room temperature reaction 1h, the colour developing of enhanced chemiluminescence method, X line egative film exposure imaging.
The result shows:
1, icariside I I is to the testing result of three kinds of melanoma cell strain cell proliferation influences
WST-8 detects cell proliferation and the comparatively advanced method of toxicity.This experimental result shows that icariside I I is external all to have shown stronger inhibitory action to three kinds of melanoma cell strains (B16, A375 and SK-MEL-2) cell proliferation, with 25-100 μ M concentration significantly (Fig. 1).
2, icariside I I is to the testing result of human melanoma cell strain A375 apoptosis influence
Adopt that Annexin V and PI are two to be dyed Flow cytometry and show that icariside I I has shown stronger cell death inducing effect to human melanoma cell strain A375, with 50-100 μ M concentration significantly (Fig. 2).
3, icariside I I is to the influence of P-P53, Caspace3 protein expression
Westernblot result shows that icariside I I can increase the phosphorylation of cyclin correlative P 53, increases cell death related protein Caspace3 protein expression, is dose-effect relationship (Fig. 3).
Embodiment 2: experiment in the body of icariside I I melanoma
Materials and methods
1, laboratory animal: the C57BL/6J mice, male, 8 ages in week, sub-cage rearing, 4 in every cage.2 of B16 tumor-bearing mices, male.20 ℃～25 ℃ of feeding environment room temperatures, relative humidity is 50%～70%, light dark period is 12h.
2, model preparation and grouping administration:
2 B16 tumor-bearing mices are taken off cervical vertebra put to death, get tumor tissue under the aseptic condition, shred, grind, sieve, preparation tumor single cell suspension, adjusting cell concentration is 1 * 10
6/ ml.In right axil subcutaneous vaccination tumor single cell suspension, every 0.2mL (contains 2 * 10 approximately with the C57BL/6J mice
5Individual tumor cell).Modeling the 8th day is divided into 3 groups with the C57BL/6J mice: dosage group, icariside I I high dose group among lotus tumor matched group, the icariside I I, 8 every group.Dosage group and high dose group give 50mg/kg and 100mg/kg lumbar injection respectively in the icariside, lotus tumor matched group be for equal volume normal saline lumbar injection, frequency injection 3 times weekly, measures major diameter a of tumor and minor axis b weekly for 3 times, and the calculating gross tumor volume, put to death in the 21st day.Gross tumor volume=0.52 * (a * b
2)
The result shows: icariside I I lumbar injection can significantly reduce the gross tumor volume of lotus B16 melanoma Mus 3 times weekly, and is dose-effect relationship (Fig. 4).
Claims (4)
1. the purposes of icariside I I in preparation melanoma medicine, described its chemical structural formula of icariside I I is as follows:
2. by the described purposes of claim 1, wherein said melanoma is boneblack melanoma or Humanmachine tumour.
3. by the described purposes of claim 1, wherein said purposes is to suppress the propagation of melanoma cell, promotes its apoptosis, increases P53 protein phosphorylation and caspace protein expression.
4. by the described purposes of claim 1, wherein said purposes is to suppress melanoma subcutaneous transplantation tumor volume.
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| CN2009101990883A CN102068448A (en) | 2009-11-19 | 2009-11-19 | Application of icariside II in preparation of anti-melanoma medicament |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103239464A (en) * | 2012-02-14 | 2013-08-14 | 复旦大学附属华山医院 | Application of icarisid II in preparation of sensitizer for chemotherapic medicine |
| CN104739822A (en) * | 2013-12-28 | 2015-07-01 | 复旦大学附属华山医院 | Application of icaritin in preparation of AMPK agonist |
| CN114315930A (en) * | 2020-09-29 | 2022-04-12 | 北京东方百奥医药开发有限公司 | Compound or pharmaceutically acceptable salt thereof, pharmaceutical composition and application |
| CN115624567A (en) * | 2022-10-19 | 2023-01-20 | 复旦大学附属华山医院 | Anticancer composition, anticancer drug and application thereof |
-
2009
- 2009-11-19 CN CN2009101990883A patent/CN102068448A/en active Pending
Non-Patent Citations (1)
| Title |
|---|
| ANLUN MA ET AL.: ""Baohuoside-1 inhibits activated T cell proliferation at G1－S phase transition"", 《TRANSPLANT IMMUNOLOGY》 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103239464A (en) * | 2012-02-14 | 2013-08-14 | 复旦大学附属华山医院 | Application of icarisid II in preparation of sensitizer for chemotherapic medicine |
| CN103239464B (en) * | 2012-02-14 | 2015-04-15 | 复旦大学附属华山医院 | Application of icarisid II in preparation of sensitizer for chemotherapic medicine |
| CN104739822A (en) * | 2013-12-28 | 2015-07-01 | 复旦大学附属华山医院 | Application of icaritin in preparation of AMPK agonist |
| CN114315930A (en) * | 2020-09-29 | 2022-04-12 | 北京东方百奥医药开发有限公司 | Compound or pharmaceutically acceptable salt thereof, pharmaceutical composition and application |
| CN114315930B (en) * | 2020-09-29 | 2024-04-05 | 北京东方百奥医药开发有限公司 | Compound or pharmaceutically acceptable salt thereof, pharmaceutical composition and application |
| CN115624567A (en) * | 2022-10-19 | 2023-01-20 | 复旦大学附属华山医院 | Anticancer composition, anticancer drug and application thereof |
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Application publication date: 20110525 |