CN102406652A - Application of forsythin in preparation of medicines for treating chronic myeloid leukemia - Google Patents

Application of forsythin in preparation of medicines for treating chronic myeloid leukemia Download PDF

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CN102406652A
CN102406652A CN2011103663332A CN201110366333A CN102406652A CN 102406652 A CN102406652 A CN 102406652A CN 2011103663332 A CN2011103663332 A CN 2011103663332A CN 201110366333 A CN201110366333 A CN 201110366333A CN 102406652 A CN102406652 A CN 102406652A
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phillyrin
leukaemia
cell
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CN102406652B (en
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尹雅玲
侯丽娟
段树鹏
朱利红
贾建伟
王苏
王倩倩
高建辉
潘国聘
赵繁荣
刘雨晴
李福林
尚校军
律海峡
李嘉
李鹏
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Xinxiang Medical University
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Abstract

The invention provides application of forsythin in preparation of medicines for treating chronic myeloid leukemia. Cell tests prove that forsythin can inhibit proliferation of K562 leukemia cell and inducing the erythroid differentiation of K562 leukemia cell.

Description

The application of phillyrin in the medicine of the anti-chronic myelocytic leukemia of preparation
Technical field
The present invention relates to the application of phillyrin, specifically, relate to the application of phillyrin in the medicine of the anti-chronic myelocytic leukemia of preparation.
Background technology
(chronic myelogenous leukemia CML) is a kind of cell strain disease that causes with the malignant change on the pluripotent stem cell level of acquired chromosomal abnormality to chronic myelocytic leukemia.Ph Chromosome t (9:22) (q34; Q11) be its distinctive cytogenetics sign, P210BCR-ABL is its molecules pathogenesis basis.How asymptomatic this disease onset is slow, in early days, mainly show as the whole body weak, become thin, hyperhidrosis, inappetence, abdominal distention, low grade fever, some cases has hemorrhage in various degree.This disease belongs to hematopoietic stem cell malignant proliferation property disease, is that hypertrophy is main with grain, does not have ill hemopoietic.Myelosis is extremely active or obvious active state, and the red ratio of grain can increase to 10 ~ 50:1, and dividing apoplexy due to endogenous wind is main with neutrophilic myelocyte, neutrophilic metamyelocyte and band-cell often.Leukocyte increases in the peripheral hemogram, generally in (100 ~ 250) * 10 9/ L, even can be up to 1000 * 10 9/ L divides apoplexy due to endogenous wind visible each stage granulocyte, is main with neutrophilic myelocyte, neutrophilic metamyelocyte and shaft-like, segmented granulocyte.
At present, the drug treatment of CML mainly comprises clinically: 1. single Drug therapy of planting, like Busulfan 4 ~ 8mg or hydroxyurea 1.5 ~ 3.0g, quiet notes, 1 time/day; 2. combined chemotherapy, (cyclophosphamide 400mg, quiet notes 1 time/day, used on the the 1st, 4 day like the COAP scheme; Vincristine 1 ~ 2mg, quiet notes 1 time/day, used on the 1st day; Cytosine arabinoside 50mg, quiet notes, per 12 hours are once, use continuously 5 ~ 9 days; Prednisone 20mg, quiet notes 1 time/day, used 5 days continuously; After the drug withdrawal 7 ~ 10 days, but alleviate fully up to symptom according to state of an illness repeated application) or the HA scheme (harringtonine 4mg/ day, quiet notes 1 time/day, used 3 days continuously; Cytosine arabinoside 100 ~ 200mg, quiet notes 1 time/day, used 7 days continuously; Have a rest after 5 ~ 7 days repeated application, totally 2 ~ 3 courses of treatment; Later harringtonine 1mg, quiet notes 1 time/day, used 20 days continuously); 3. interferon (IFN) treatment is like IFN-α-2b, 2 * 10 6U/m 2~ 5 * 10 6U/m 2Or with 2 * 10 7U/m 2, quiet notes, 1 time/day.
The therapeutic goal of traditional chronic myelocytic leukemia is conceived to killer cell and then suppresses propagation.This therapy also can produce inhibition to normal hematopoietic cell usually when killing and wounding the leukaemia.From the mid-80 in last century, the all-trans-retinoic acid that Chinese scholar is carried out is effectively treated the research of acute promyelocytic leukemia, and the induction-differential therapy of tumor is become a reality.Up to now, found that many pharmacological active substancies such as Buddhist ripple ester, sodium butyrate, 5-azepine deoxycytidine etc. all can break up by inducing cancer cell, comprising leukaemia and some solid tumor cell.
Phillyrin is extracted from the dry fruit of Oleaceae plant Fructus Forsythiae [Forsythia suspensa (Thunb.) Vahl], and molecular formula is C 27H 34O 11, molecular weight is 534.55.We discover that phillyrin can suppress the K562 Leukemia Cell Proliferation, induce K562 leukaemia to break up to red system.
Summary of the invention
The object of the present invention is to provide the new purposes of phillyrin aspect the medicine of the anti-chronic myelocytic leukemia of preparation.
Specifically, the present invention relates to the application of phillyrin in the medicine of the anti-chronic myelocytic leukemia of preparation.
Wherein, described application is characterized in that suppressing the K562 Leukemia Cell Proliferation, induces K562 leukaemia to break up to red system.
Phillyrin of the present invention is extracted from the dry fruit of Oleaceae plant Fructus Forsythiae [Forsythia suspensa (Thunb.) Vahl].
Medicine according to the invention is meant with the phillyrin to be main active, and acceptable preparation in the medical treatment of processing with pharmaceutically suitable carrier is like injection, tablet, pill or capsule etc.
Medicine according to the invention, its consumption are in phillyrin: adult, 40 ~ 50mg/ time, 2 times/day; The child 10 ~ 15mg/kg time, 2 times/day, can reach the effect that suppresses Leukemia Cell Proliferation and induce differentiation.Need follow the doctor's advice when especially, the anemia of pregnant woman waits special population to use.
Phillyrin according to the invention can directly obtain through commercially available, also can obtain through plant extract.
The present invention is through 2, two (2-methoxyl group-4-nitro-5 sulfo group the benzene)-5-of 3-[(anilino-) carbonyl)]-dihydro-tetrazole hydrogen (XTT) evidence phillyrin can suppress the K562 Leukemia Cell Proliferation.
The present invention has proved that through colony-forming test phillyrin can suppress the K562 Leukemia Cell Proliferation.
The present invention through the morphology evidence phillyrin can suppress the K562 Leukemia Cell Proliferation, and the trend of the differentiation of inducing is arranged.
The present invention detects K562 leukaemia's cycle through flow cytometer, find phillyrin to K562 leukaemia's inhibited proliferation possibly be since with K562 leukaemia's Cycle Arrest due to the S phase.
The present invention has proved that through the hemoglobin benzidine staining phillyrin has the ability of K562 leukaemia to the terminal cell differentiation that produces hemoglobin of inducing.
The present invention detects CD71 surface markers on K562 leukaemia's after birth through flow cytometer, has proved that phillyrin can induce K562 leukaemia to break up to red system.
Beneficial effect of the present invention is:
1, the invention provides the new purposes of phillyrin, it has the good restraining proliferation function to K562 leukaemia, and can induce K562 leukaemia to break up to red system.
2, the phillyrin of the present invention's employing is extracted from pure natural plant, and its chemical constitution and content are clear and definite, and is quality controllable, meets the requirement of international market third generation tcm product, and vast market prospect is arranged.If study successfully, with the international status that improves China's Chinese medicine, to ensureing that human health contributes.
Description of drawings
Fig. 1 phillyrin is to the morphologic influence of K562 leukaemia (* 400).Result of the test shows that K562 leukaemia's cell volume after phillyrin is handled becomes big, and the caryoplasm ratio reduces; The nuclear incisura is arranged, and nuclear chromatin concentrates, chap, and the part entoblast is apparent in view; Kytoplasm increases, understain, examines all kytoplasms and olistherozone occurs, performance differentiation to a certain degree.
Fig. 2 phillyrin is to the influence of hemoglobin benzidine staining in the K562 leukaemia.Result of the test shows that phillyrin has induces the ability of K562 leukaemia to the terminal cell differentiation that produces hemoglobin, and has dose dependent.
Fig. 3 phillyrin is to the influence of K562 leukaemia's film CD71 surface markers.Result of the test shows that phillyrin can induce K562 leukaemia to break up to red system, and has dose dependent.
The specific embodiment
Below come further to set forth the new purposes and the beneficial effect thereof of phillyrin of the present invention through embodiment and Test Example, but be not used for limiting scope of the present invention.
Test Example 1:Phillyrin is to K562 leukaemia's inhibited proliferation.
1, medicine: phillyrin (the emerging commerce and trade company limited of Shanghai section), XTT (U.S. Sigma company).
2, instrument: ELX-800 ELIASA (U.S. Bio-Tek company).
3, cell: K562 leukaemia (cell resource center of Shanghai Sheng Ke institute of the Chinese Academy of Sciences).
4, experiment is divided into groups: (1) blank group; (2) 10 -5Mol/L hydroxyurea matched group; (3) 10 -7Mol/L phillyrin group; (4) 10 -6Mol/L phillyrin group; (5) 10 -5Mol/L phillyrin group; (6) 10 -4Mol/L phillyrin group.
5, experiment content: XTT test.
6, statistical method: adopt SPSS13.0 software; Experimental data is with (
Figure 2011103663332100002DEST_PATH_IMAGE001
± s) expression; Relatively adopt the repeated measure variance analysis between the many groups of cell counting; Relatively adopt one factor analysis of variance between many means, < 0.05 is that difference has statistical significance with P.
7, result: 10 -4Mol/L phillyrin can significantly suppress the K562 Leukemia Cell Proliferation, compares with the blank group, has significant difference (P<0.05); With 10 -5Mol/L hydroxyurea matched group is compared, there was no significant difference (P＞0.05); Phillyrin suppresses the effect of K562 Leukemia Cell Proliferation and has tangible dose dependent.(table 1)
Table 1 phillyrin is to the influence of K562 leukaemia XTT
Group OD 570
The blank group 1.18±0.25 #
10 -5Mol/L hydroxyurea matched group 0.45±0.09 *
10 -7Mol/L phillyrin group 1.11±0.22 #
10 -6Mol/L phillyrin group 0.97±0.18 *#
10 -5Mol/L phillyrin group 0.72±0.15 *#
10 -4Mol/L phillyrin group 0.42±0.11 *
Annotate: compare * with the blank group<0.05; With 10 -5Mol/L hydroxyurea matched group compares, #<0.05.
Test Example 2:Phillyrin is to K562 leukaemia's inhibited proliferation.
1, medicine: phillyrin (the emerging commerce and trade company limited of Shanghai section).
2, cell: with Test Example 1.
3, experiment is divided into groups: with Test Example 1.
4, experiment content: colony forms inhibition test.
5, statistical method: with Test Example 1.
6, result: 10 -4Mol/L phillyrin can significantly suppress K562 leukaemia's colony and form, and compares with the blank group, has significant difference (P<0.05); With 10 -5Mol/L hydroxyurea matched group is compared, there was no significant difference (P＞0.05); The effect that phillyrin suppresses the formation of K562 leukaemia's colony has tangible dose dependent.(table 2)
Table 2 phillyrin is to the influence of K562 leukaemia's colony generation rate
Group The colony number (individual/0.5ml)
The blank group 410.22±30.26 #
10 -5Mol/L hydroxyurea matched group 115.36±18.25 *
10 -7Mol/L phillyrin group 402.65±32.97 #
10 -6Mol/L phillyrin group 326.57±28.61 *#
10 -5Mol/L phillyrin group 268.11±22.37 *#
10 -4Mol/L phillyrin group 110.23±16.25 *
Annotate: compare * with the blank group<0.05; With 10 -5Mol/L hydroxyurea matched group compares, #<0.05.
Test Example 3:Phillyrin is to the morphologic influence of K562 leukaemia.
1, medicine: with Test Example 2.
2, cell: with Test Example 1.
3, experiment is divided into groups: with Test Example 1.
4, experiment content: the om observation K562 leukaemia Wright-Giemsa form that dyes.
5, result: K562 leukaemia's endochylema navy blue of blank group, nuclear is big, and is rounded or oval, and chromatin is loose careful, visible 1 ~ 3 kernel, the caryoplasm ratio is big; K562 leukaemia's cell volume after phillyrin is handled becomes big, and the caryoplasm ratio reduces, and the nuclear incisura is arranged, and nuclear chromatin concentrates, chap, and the part entoblast is apparent in view, and kytoplasm increases, understain, examines all kytoplasms and olistherozone occurs, performance differentiation to a certain degree.(Fig. 1)
Test Example 4:Phillyrin is to the influence in K562 leukaemia's cycle.
1, medicine: with Test Example 2.
2, cell: with Test Example 1.
3, instrument: FACScaliber flow cytometer (U.S. Becton-Dickinson company).
4, experiment is divided into groups: with Test Example 1.
5, experiment content: cell cycle detects.
6, statistical method: with Test Example 1.
7, result: 10 -4Mol/L phillyrin component is distributed in cell and the apparent in view increase (P of blank group of S phase<0.05), compare with the hydroxyurea matched group, there was no significant difference (P＞0.05), and be distributed in cell number and the apparent in view minimizing (P of blank group of G1 phase and G2 phase<0.05).It is thus clear that, phillyrin K562 leukaemia's inhibitory action is likely since with cell cycle arrest due to the S phase, and have dose dependent.(table 3)
Table 3 phillyrin to the influence in K562 leukaemia's cycle (
Figure 409558DEST_PATH_IMAGE001
± s, n=3)
Annotate: compare * with the blank group<0.05; With 10 -5Mol/L hydroxyurea matched group compares, #<0.05.
Test Example 5:Phillyrin is to the influence of hemoglobin benzidine staining in the K562 leukaemia.
1, medicine: with Test Example 2.
2, reagent: benzidine dihydrochloride (U.S. Signa company).
3, instrument: with Test Example 1.
4, cell: with Test Example 1.
5, experiment is divided into groups: with Test Example 1.
6, experiment content: benzidine staining.
7, statistical method: with Test Example 1.
8, result: 10 -4K562 leukaemia's benzidine staining A value of mol/L phillyrin group obviously raises, and with the blank group notable difference (P is arranged relatively<0.05), compare there was no significant difference (P＞0.05) with the hydroxyurea matched group; Explain that phillyrin has the ability of inducing K562 leukaemia to break up to the terminal cell that produces hemoglobin, and have dose dependent.(Fig. 2)
Test Example 6:Phillyrin is to the influence of K562 leukaemia's film CD71 surface markers.
1, medicine: with Test Example 2.
2, cell: with Test Example 1.
3, reagent: the mouse-anti people CD71 monoclonal antibody of FITC labelling (Shenzhen brilliant U.S. biotech firm).
4, instrument: with Test Example 4.
5, experiment is divided into groups: with Test Example 1.
6, experiment content: benzidine staining.
7, result: 10 -4The mol/L phillyrin acted on K562 leukaemia after 4 days, and the CD71 surface markers is expressed the positive on 83% the cell membrane, with the blank group notable difference (P is arranged relatively<0.05), compare there was no significant difference (P＞0.05) with the hydroxyurea matched group; Explain that phillyrin can induce K562 leukaemia to break up to red system, and have dose dependent.(Fig. 3)

Claims (4)

1. the application of phillyrin in the medicine of the anti-chronic myelocytic leukemia of preparation.
2. phillyrin according to claim 1 is characterized in that, is extracted from the dry fruit of Oleaceae plant Fructus Forsythiae [Forsythia suspensa (Thunb.) Vahl].
3. phillyrin according to claim 1 is characterized in that molecular formula: C 27H 34O 11, molecular weight: 534.55, CAS accession number: 487-41-2, chemical name: b-D-Glucopyranoside, [4-(3 for 4-; 4-dimethoxyphenyl) tetrahydro-1H, 3H-furo [3,4-c] furan-1-yl]-2-methoxyphenyl, [1S-(1a; 3aa, 4b, 6aa)]-, structural formula:
。
4. application according to claim 1 is characterized in that, suppresses the K562 Leukemia Cell Proliferation, induces K562 leukaemia to break up to red system.
CN 201110366333 2011-11-18 2011-11-18 Application of forsythin in preparation of medicines for treating chronic myeloid leukemia Expired - Fee Related CN102406652B (en)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102784160A (en) * 2012-09-03 2012-11-21 苏州大学 Application of forsythin to preparation of medicine for improving cognitive function and treating Alzheimer's diseases
CN105030948A (en) * 2015-08-31 2015-11-11 陕西郝其军制药股份有限公司 Drug combination and new application of preparation thereof

Citations (2)

* Cited by examiner, † Cited by third party
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CN1602855A (en) * 2004-08-05 2005-04-06 陕西师范大学 Application of forsythin in the process for preparing adiposis treating oral medicine and healthy food
CN1947747A (en) * 2005-10-10 2007-04-18 黄振华 Traditional Chinese medicine composition contg. luteolin and capsule of sweeping forsythia and its prepn. method and use

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1602855A (en) * 2004-08-05 2005-04-06 陕西师范大学 Application of forsythin in the process for preparing adiposis treating oral medicine and healthy food
CN1947747A (en) * 2005-10-10 2007-04-18 黄振华 Traditional Chinese medicine composition contg. luteolin and capsule of sweeping forsythia and its prepn. method and use

Non-Patent Citations (2)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102784160A (en) * 2012-09-03 2012-11-21 苏州大学 Application of forsythin to preparation of medicine for improving cognitive function and treating Alzheimer's diseases
CN105030948A (en) * 2015-08-31 2015-11-11 陕西郝其军制药股份有限公司 Drug combination and new application of preparation thereof
CN105030948B (en) * 2015-08-31 2019-05-17 陕西郝其军制药股份有限公司 A kind of new application of pharmaceutical composition and its preparation

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