CN104208061B - The medical usage of berberinc derivate - Google Patents
The medical usage of berberinc derivate Download PDFInfo
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Abstract
The present invention provides the application of aspect in preparation treatment glioblastoma tumor medicine or Mitochondrially targeted drug-supplying system of the compound or its salt of formula (I):Formula (I), wherein, R is C10‑C18Alkyl, or R is benzyl.The present invention is found through experiments, and compared with the control, the berberinc derivate of the present invention can not only more effectively suppress the propagation of glioma cell, more can simultaneously effective suppress migration and the invasive ability of cell.Additionally, these compounds can navigate in mitochondrion well, therefore, it is possible to as Mitochondrially targeted drug-supplying system.
Description
Technical field
The present invention relates to the derivant of the substituted chain alkyl of the new application of berberinc derivate, particularly C9-O or the benzyl new application in terms for the treatment of glioblastoma.
Background technology
Glioblastoma (Malignant Glioma) is modal primary malignant neoplasm in central nervous system, and sickness rate accounts for the 46% of intracranial tumor.Operation is first-selected Therapeutic Method, but owing to it is infiltrative growth, is difficult to excise without obvious boundary, operation with normal cerebral tissue, and Postoperative recurrent rate is up to 96%.Even if being aided with radiation and chemotherapy, postoperative mean survival time (MST), is also less than 14 months, poor prognosis.The property of can not be cured of glioblastoma migrates by force with it and invasive ability is closely related.Therefore, the migration of suppression glioma cell is the most crucial to the treatment of glioblastoma with invasion and attack.But, existing therapies means can not suppress migration and the invasion and attack of glioblastoma effectively, causes therapeutic effect the best, and also there is many defects such as neurotoxicity, hematotoxicity, drug resistance.
Additionally, including tumor, the pathological process of multiple disease is relevant with mitochondrial injury.Structurally and functionally there is larger difference in tumor cell and Normocellular mitochondrion, the exception of the intracellular biochemical event directly related with mitochondrion, the such as change of carbohydrate metabolism pattern, ATP dyspoiesis, calcium ion accumulation, oxidative stress etc. are all the major reason of tumor development in functional disorder in various degree.Meanwhile, in tumor cell, mtDNA sudden change and excess ROS have been in the news promotion Nasopharyngeal neoplasms.This makes drug targeting in the mitochondrion of tumor cell, and is selectively applied to tumor cell by mitochondrion and is possibly realized, and this invasive tumor of height this kind of for targeted therapy glioblastoma is significant.
Berberine (Berberine) is a kind of isoquinoline alkaloid extracting isolated from the Chinese medicine materials such as Rhizoma Coptidis, and oral administration safety is high, takes the most in a large number without blood, cardiovascular and Liver and kidney toxicity.Research shows, berberine can suppress migration and the invasion and attack of hepatocarcinoma, human tongue carcinoma, melanoma, breast carcinoma, bladder cancer etc., but has no the report on glioma migration Yu the impact of invasion and attack.On the other hand, although berberine has the inhibitory activity to some tumor, but its drug effect is not ideal.
The present invention intends, the chemotherapeutics of low toxicity a kind of safely, effectively in exploitation, and this chemotherapeutics can effectively suppress migration and the invasion and attack of Malignant glioma cells.
Summary of the invention
For solving the problems referred to above, the present invention provides a kind of new therapeutic scheme for glioblastoma, and the unexpected inhibition of Malignant glioma cells is realized by it based on berberinc derivate.
According to the first aspect of the invention, the present invention provides the application in terms of preparation treatment glioblastoma tumor medicine of the compound or its salt of formula (I):
,
Wherein, R is C10-C18Alkyl, or R is benzyl.Described C10-C18Alkyl includes C10-C18Alkyl or their isomer.
In a preferred embodiment, R is C10-C18Alkyl, such as decane base, n-undecane base, dodecyl, n-tridecane base, n-tetradecane base, Pentadecane base, n-hexadecyl, n-heptadecane base, n-octadecane base.More preferably C10-C16Alkyl.It is highly preferred that R is C12-C16Alkyl.It is highly preferred that R is dodecyl or n-hexadecyl.
In a preferred embodiment, described salt is selected from hydrobromate, hydriodate, hydrofluoride, hydrochlorate, sulfate, nitrate, phosphate, citrate, acetate and lactate, preferably hydrobromate, hydrochlorate or sulfate.
According to the preferred embodiment of the present invention, described compound is selected from: 9-O-decane base berberine, 9-O-dodecyl berberine, 9-O-n-hexadecyl berberine, 9-O-n-octadecane base berberine, and 9-O-benzyl berberine.
According to an embodiment of the invention, described pharmaceutical pack is extra to the treatment effective composition of glioblastoma containing at least one, is used in combination with the derivant with the present invention.According to an embodiment of the invention, described pharmaceutical pack contains pharmaceutical excipient, so that described medicine is made suitable dosage form.
Inventor finds, the berberinc derivate of the substituted chain alkyl of 9-O or benzyl is better than other berberinc derivates and berberine itself unexpectedly for growth inhibited, inhibition of metastasis and the invasion and attack suppression of Malignant glioma cells.The present invention is found through experiments, and compared with the control, berberinc derivate can not only more effectively suppress the propagation of C 6 Cell of Glioma, more can simultaneously effective suppress migration and the invasive ability of cell.Possible reason is, when lipophilic group (chain alkyl or benzyl) is combined with ehter bond with parent drug berberine, improve the fat-soluble of compound, by improving transmembrane transport, and compound is transported to diseased region and intracellular through biomembrane effectively, thus enhance drug effect.
According to the second aspect of the invention, the present invention provides the application in preparing Mitochondrially targeted drug-supplying system of the compound or its salt of formula (I):
,
R is C10-C18Alkyl or benzyl.Described C10-C18Alkyl includes C10-C18Alkyl or their isomer.
In a preferred embodiment, R is C10-C18Alkyl, such as decane base, n-undecane base, dodecyl, n-tridecane base, n-tetradecane base, Pentadecane base, n-hexadecyl, n-heptadecane base, n-octadecane base.More preferably C10-C16Alkyl.It is highly preferred that R is C12-C16Alkyl.It is highly preferred that R is dodecyl or n-hexadecyl.
According to the preferred embodiment of the present invention, described compound is selected from: 9-O-ten alkyl berberine, 9-O-dodecyl berberine, 9-O-cetyl berberine, 9-O-octadecyl berberine, and 9-O-benzyl berberine.
In a preferred embodiment, described salt is selected from hydrobromate, hydriodate, hydrofluoride, hydrochlorate, sulfate, nitrate, phosphate, citrate, acetate and lactate, preferably hydrobromate, hydrochlorate or sulfate.
Described Mitochondrially targeted drug-supplying system comprises at least one extra ingredient, and this ingredient generally self is difficult to enter mitochondrion, and with the derivant of the present invention, harmful phase interaction does not occur.This extra ingredient can be positioned in mitochondrion under the effect of the derivant of the present invention, thus plays a role.
According to the third aspect of the present invention, the present invention provides a kind of method treating glioblastoma, and the method includes the compound or its salt of the formula (I) of therapeutically effective amount is applied to patient in need.
In the present invention, C10-C18Alkyl refers to the straight or branched alkyl with 10 to 18 carbon atoms, similarly, C12-C16Alkyl refers to the straight or branched alkyl with 12 to 16 carbon atoms.
Accompanying drawing explanation
Fig. 1 shows that the berberine of variable concentrations and derivant thereof act on the change of cells survival rate after C6 cell 24 h.
Fig. 2 shows that the berberinc derivate of variable concentrations acts on the change of cells survival rate after C6 cell 24 h, 48 h and 72 h, (A) derivant 9-O-dodecyl berberine, it is called for short B3, (B) derivant 9-O-cetyl berberine, it is called for short B4, (C) derivant 9-O-octadecyl berberine, is called for short B5, (D) derivant 9-O-benzyl berberine, is called for short B6.
Fig. 3 shows berberine and derivant C6 cell scratch experiment result thereof, (A) the cell cut of matched group, berberine group and berberinc derivate group cell migration situation after 0h and 24 h, (B) relative mobility between each group compares, and compares, and * represents P
<0.05；* represents P
<0.01；* * represents P
<0.001.B3 represents 9-O-dodecyl berberine；B4 represents 9-O-cetyl berberine；B5 represents 9-O-octadecyl berberine；B6 represents 9-O-benzyl berberine.
Fig. 4 shows that berberine and derivant C6 cell Transwell thereof migrate experimental result, (A) the cell migration situation of matched group, berberine group and berberinc derivate group, (B) relative mobility between each group compares, and compares, and * represents P < 0.05；* represents P < 0.01；* * represents P < 0.001.B3 represents 9-O-dodecyl berberine；B4 represents 9-O-cetyl berberine；B5 represents 9-O-octadecyl berberine；B6 represents 9-O-benzyl berberine.
Fig. 5 shows berberine and derivant C6 cell Transwell Matrigel result thereof, and the invasion and attack of cell are suppressed situation by (A) matched group, berberine group and berberinc derivate group；(B) relative mobility between each group compares, and compares, and * represents P < 0.05；* represents P < 0.01；* * represents P < 0.001.B3 represents 9-O-dodecyl berberine；B4 represents 9-O-cetyl berberine；B5 represents 9-O-octadecyl berberine；B6 represents 9-O-benzyl berberine.
Fig. 6 is the laser co-focusing observed result of berberine lipophilic derivatives mitochondrion location in C6 (A) and U87 (B) glioma cell.Mitotracker represents the mitochondrion of mitotracker green labelling, in green fluorescence；Medicine represents berberine and lipophilic derivatives thereof, in yellow fluorescence；Superposition represents the superposition of two above image.B3 represents 9-O-dodecyl berberine；B4 represents 9-O-cetyl berberine.
Detailed description of the invention
The synthesis of berberinc derivate and qualification
Berberine hydrochloride is purchased from Xi'an Caulis et Folium Polygalae Tenuifoliae plant Science and Technology Ltd., purity 97%；Bromododecane, bromohexadecane, bromo-octadecane, cylite are analytical pure, are purchased from Aladdin reagent company limited；DMF (DMF) is analytical pure, processes by molecular sieve drying, is purchased from Tianjin Fu Yu Fine Chemical Co., Ltd；Chromatography neutral alumina is traditional Chinese medicines group product；DMEM culture medium (R10-013-cv), hyclone (FBS), trypsin, penicillin and streptomycin are all purchased from cellgro company of the U.S.；Tetramethyl azo azoles salt (MTT) and dimethyl sulfoxide (DMSO) are all purchased from Sigma Co., USA；4% paraformaldehyde is purchased from Guangzhou Jing Xin Science and Technology Ltd.；Experimental water is ultra-pure water.Rat glioma C 6 cells, people's glioma U87 cell are from U.S.'s ATCC cell bank.Millicell Cell Culture Inserts (8.0 μm PET) is U.S.'s Millipore Products.AvanceⅢ400MHz nuclear magnetic resonance spectrometer is U.S.'s Bruker Products；Ultra Performance Liquid Chromatography tandem mass spectrometer UPLC-MS/MS(TSQ Quantum Access Max) it is U.S. Thermo
Scientific product；High speed refrigerated centrifuge (LEGEND MICRO 17R) is U.S.'s Thermo scientific Products；Microplate reader (Microplate
Reader) it is U.S.'s BIO-RAD Products；It is inverted research grade microscope (IX
71) it is Japan's OLYMPUS Products；Water-jacket typ CO2Incubator (HEPA class100) is U.S. Thermo
Scientific Products.
One exemplary synthetic pathways of berberinc derivate of the present invention is as follows:
1.1
Berberrubine
(Berberrubine,
B2)
Synthesis and qualification
Use high-temperature cracking method synthetic intermediate B2。
Take dry berberine hydrochloride 10 g(0.0269 mol), at N2Protect lower 190 DEG C of Pintsch process 5 h.Being cooled to room temperature, acetone 50 mL cleans, and obtains red crystals 7.48 g, productivity 87%.1H NMR (400 MHz, CD3OD) δ: 9.22 (s, 1H), 7.96 (s, 1H), 7.48 (d, J = 8.2 Hz,
1H), 7.39 (s, 1H), 6.83 (d, J = 8.3 Hz, 1H), 6.81 (s, 1H), 6.02 (s, 2H),
4.52-4.62 (t, J = 6.3 Hz, 2H), 3.86 (s, 3H), 3.05-3.14 (t, J =
6.3 Hz, 2H); 13C NMR (101 MHz, CD3OD) δ: 151.11, 150.85, 149.47, 147.31, 135.54, 133.74, 130.66,
124.21, 122.90, 121.71, 119.63, 109.20, 108.17, 105.81, 103.25, 56.79, 55.53,
28.96; ESI-MS m/z: 322 [M+H]+。
Berberinc derivate
B3, B4, B5
,
B6
With
B12
Synthesis and qualification
WillB2React with corresponding bromo-derivative and respectively obtainB3,
B4, B5, B6 With B12。
TakeB2 0.32 g(1
Mmol) it is dissolved in 5 mL DMF, at N2Under protection, 80 DEG C are reacted 2h ~ 24h to the 4 corresponding bromo-derivatives of mmol, filter, and take precipitation, dry method loading, neutral alumina column purification (chloroform-methanol 200～100:1), obtain yellow crystal respectivelyB3, B4,, B5 , B6 With B12 。Productivity 70% ~ 80%.
9-O-dodecyl berberine (B3)1H NMR
(400 MHz, DMSO) δ: 9.76 (s, 1H),
8.95 (s, 1H), 8.19 (d, J = 9.1 Hz, 1H), 7.99 (d, J = 9.1 Hz, 1H),
7.80 (s, 1H), 7.09 (s, 1H), 6.17 (s, 2H), 4.91 (t, J = 6.7 Hz, 2H), 4.28
(t, J = 6.7 Hz, 2H), 4.05 (s, 3H), 3.24 (t, J = 6.7 Hz, 2H),
1.81-1.94 (m, 2H), 1.42-1.52 (m, 2H), 1.39-1.22 (m, 16H), 0.85 (t, J =
6.7 Hz, 3H); 13C NMR (101 MHz, DMSO) δ:
150.34, 149.76, 147.62, 145.20, 142.84, 137.36, 133.01, 130.58, 126.62, 123.26,
121.61, 120.37, 120.16, 108.35, 105.38, 102.03, 74.22, 57.00, 55.30, 31.23,
29.43, 28.97, 28.77, 28.65, 26.30, 25.21, 22.03, 13.87; ESI-MS m/z: 490
[M-Br]+。
9-O-cetyl berberine (B4)1H NMR
(400 MHz, DMSO) δ: 9.75 (s, 1H)
8.94 (s, 1H), 8.20 (d, J = 8.9 Hz, 1H), 8.00 (d, J = 8.9 Hz, 1H),
7.80 (s, 1H), 7.09 (s, 1H), 6.18 (s, 2H), 4.95 (t, J = 6.7 Hz, 2H), 4.29
(t, J = 6.7 Hz, 2H), 4.05 (s, 3H), 3.23 (t, J = 6.7 Hz, 2H),
1.87-1.96 (m, 2H), 1.48-1.58 (m, 2H), 1.24 (s, 24 H), 0.85 (t, J = 3.4 Hz,
3H);13C NMR (101 MHz, DMSO) δ:
150.36, 149.78, 147.65, 145.23, 142.86, 137.40, 133.02, 130.62, 126.68, 123.24,
121.63, 120.40, 120.18, 108.36, 105.39, 102.05, 74.21, 57.01, 55.29, 31.23,
29.43, 28.99, 28.78, 28.64, 26.31, 25.22, 22.03, 13.87; ESI-MS m/z: 546
[M-Br]+。
9-O-octadecyl berberine (B5)
1H NMR (400 MHz, DMSO) δ: 9.75 (s, 1H), 8.94 (s, 1H), 8.19 (d, J = 9.0 Hz,
1H), 8.00 (d, J = 9.0 Hz, 1H), 7.80 (s, 1H), 7.09 (s, 1H), 6.17 (s, 2H),
4.95 (t, J = 6.4 Hz, 2H), 4.28 (t, J = 6.4 Hz, 2H), 4.05 (s, 3H),
3.23 (t, J = 5.3 Hz, 2H), 1.83-1.92 (m, 2H), 1.42-1.52 (m, 2H),
1.39-1.25 (m, 28H), 0.86 (t, J = 4.9 Hz, 3H); 13C NMR (101
MHz, DMSO) δ: 150.37, 149.80, 147.66, 145.22,
142.87, 137.44, 133.03, 130.63, 126.70, 123.26, 121.65, 120.40, 120.18, 108.37,
105.39, 102.04, 74.22, 57.01, 55.30, 31.21, 29.41, 28.95, 28.75, 28.62, 26.31,
25.21, 22.01, 13.87; ESI-MS m/z: 574 [M-Br]+。
9-O-benzyl berberine (B6)1H NMR
(400 MHz, DMSO) δ: 9.73 (s, 1H),
8.93 (s, 1H), 8.21 (d, J = 9.1 Hz, 1H), 8.00 (d, J = 9.0 Hz, 1H),
7.78 (s, 1H), 7.59 (d, J = 7.3 Hz, 2H), 7.38 (dt, J = 8.5, 5.1
Hz, 3H), 7.08 (s, 1H), 6.17 (s, 2H), 5.36 (s, 2H), 4.91 (t, J = 6.7 Hz,
2H), 4.08 (s, 3H), 3.21 (t, J = 6.7 Hz, 2H); 13C NMR (101
MHz, DMSO) δ: 150.66, 149.78, 147.63, 145.24,
141.95, 137.30, 136.40, 132.89, 130.57, 128.75, 128.37, 128.31, 126.49, 123.71,
121.77, 120.32, 120.15, 108.36, 105.39, 102.04, 75.32, 57.03, 55.33, 26.31;
ESI-MS m/z: 412 [M-Br]+。
9-O-ten alkyl berberine (B12)1H NMR
(400 MHz, DMSO) δ 9.74 (s, 1H),
8.93 (s, 1H), 8.18 (d, J = 8.8 Hz, 1H), 7.98 (d, J = 9.1 Hz, 1H),
7.78 (s, 1H), 7.08 (s, 1H), 6.16 (s, 2H), 4.94 (t, J = 6.3 Hz, 2H), 4.26
(t, J = 6.7 Hz, 2H), 4.04 (s, 3H), 3.20 (s, 3H), 1.91 – 1.81 (m, 2H), 1.45 (d, J = 7.3 Hz, 2H), 1.24 (s,
12H), 0.84 (d, J = 6.8 Hz, 3H); 13C NMR (101 MHz, DMSO) δ: 150.34, 149.72, 147.59, 145.23, 142.82, 137.32, 132.99,
130.57, 126.57, 123.27, 121.59, 120.38, 120.19, 108.33, 105.39, 102.03, 74.21,
56.99, 55.24, 31.26, 29.45, 28.96, 28.81, 28.67, 26.29, 25.23, 22.05, 13.90;
ESI-MS m/z: 462 [M-Br]+。
2.
The functional verification of berberinc derivate
2.1
Cell is cultivated
C6 and U87 glioma cell is incubated at the DMEM high glucose medium of 10% hyclone (FBS), at 5% CO2, cultivate under conditions of 37 DEG C.Culture fluid, Secondary Culture is changed every 48 h.
The survival rate of method detection cell
If matched group and 5 variable concentrations medicine groups, the often multiple hole of group 3.Take the logarithm the C6 cell dissociation of trophophase, counting, be inoculated in 96 orifice plates by cell number 10000/ hole, be incubated at 37 DEG C, 5% CO2After incubator 24 h, add the drug solution of variable concentrations.After continuing to cultivate 24 h, 48 h, 72 h, every hole adds tetrazolium salts solution (MTT, 5 mg/mL)
20 μ L continue to cultivate 4 h, inhale and abandon supernatant, and every hole adds DMSO 100 μ L, shakes 10 min, microplate reader measures the absorbance (A) of 570 nm.Cell survival rate (%)=AMedicine group/ AMatched group× 100%.Result is as depicted in figs. 1 and 2.As seen from Figure 1, berberine and derivant thereof are dose dependent to the inhibited proliferation of C6 cell, between variable concentrations group (0,5,10,20,40 μMs) cell survival rate difference have statistical significance (P<0.05).Compared with matched group, berberine concentration more than 20 μMs time present obvious inhibited proliferation (P< 0.05).The inhibitory action relatively berberine of berberinc derivate (B3, B4, B5 and B6) cell proliferation notable (P< 0.05).Wherein, the inhibited proliferation of B3 is the strongest, and 5 μMs i.e. proliferation activity to cell produces and significantly affects, and the cells survival rate of each concentration all significantly lower than berberine and other derivants (P< 0.05).Fig. 2 is that B3, B4, B5 and the B6 of variable concentrations acts on the change of cells survival rate after C6 cell 24 h, 48 h and 72 h.Berberinc derivate to the inhibitory action of C6 cell proliferation be obvious dosage and time dependence (P< 0.05).
Cell scratch test
Take the logarithm the C6 cell of trophophase be inoculated in 6 orifice plates, it is placed in incubator, after cell covers with monolayer about 90%, cell cut is done equably with 20 mL rifle heads of sterilizing, phosphate buffer (Phosphate Buffer Solution, PBS) rinses cell debris 3 times, is replaced by serum-free medium, if matched group and medicine group, medicine group culture fluid drug containing concentration is 5
mmol/L.By the extent of migration of inverted microscope (× 40) observation of cell and take pictures after 24 h.By Image J2x (2.1.4.7 version) software measurement cell migration distance.According to cell relative migration distance=(0 h scratch width-24 h scratch width)/0 h scratch width × 100%, calculate the relative migration distance of each group, then with the relative migration distance of matched group for 100%, calculate the relative mobility of medicine group.Result is as shown in Figure 3.Fig. 3 A shows, the cell that the cell cut of matched group and berberine group is almost migrated after 24 h is covered, and area coverage reaches more than 90%；And the scored area of each derivant group is the most clearly visible.Relative mobility (Fig. 3 B) between more each group, has significant difference, and derivant is significantly stronger than crude drug berberine (P < 0.05) to the inhibitory action migrated between medicine group and matched group.Wherein, B3 is the strongest to the inhibition of metastasis effect of C6 cell, and relative mobility (13.75% ± 4.86%) is significantly lower than other derivant groups (P < 0.05).The relative mobility of B4 group is 23.56% ± 8.67%, and the inhibitory action of cell migration is only second to B3, and is better than B5 and B6(relative mobility and is respectively 35.46% ± 2.33% and 46.89% ± 10.56%).
Migration test
Trypsinization also collects cell, and the DMEM culture fluid containing 10% FBS washs 3 times, the resuspended (cell number 2.5 × 10 of DMEM culture fluid of serum-free5/ mL).It is uniformly added into the 100 supreme rooms of μ L cell suspension, is simultaneously introduced the drug solution of suitable concentration；Lower room adds containing 10%
The DMEM culture fluid 600 μ L of FBS.Meanwhile, MTT experiment is carried out with detection cell quantity change with 96 orifice plates of same cell number.37 DEG C, 5% CO2After cultivating 24 h, room in taking-up, wipe upper ventricular cell with cotton swab, 4% paraformaldehyde fixes 30
Min, 0.2% crystal violet dye liquor dyeing 10
Min, distillation washing three times, randomly select 9 visuals field under microscope (× 200) and take the photograph sheet and count；Often organizing and parallel set 3 apertures, experiment is repeated 3 times.Finally, the mobility evaluation of the transfer ability of cell, total cell number × 100% of MTT under mobility=migrating cell number/isoconcentration.Each group mobility and the ratio i.e. relative mobility of matched group mobility.Result is as shown in Figure 4.Fig. 4 A shows,B3WithB4Group migrate to the cell number of lower room minimum (P< 0.001).
Invasion and attack test
Matrigel (BS Biosciences) 5 mL are placed in 4 DEG C and overnight thaw, dilute according to DMEM:Matrigel=3:1 with the serum-free DMEM culture fluid of 4 DEG C of pre-coolings, the Matrigel 50 μ L taking dilution adds room on the Transwell of pre-cooling, and 37 DEG C of incubation 4 h make gel formation.Hereinafter operation is same2.4It is described that (upper room inoculating cell amount is 5 × 105 /100
μL).Finally, the invasive ability of cell invasion and attack rate is evaluated, total cell number × 100% of MTT under invasion and attack rate=invasion and attack cell number/isoconcentration.Each invasion and attack rate ratio with matched group invasion and attack rate of organizing is i.e. relative to invasion and attack rate.
Result is as shown in Figure 5.After drug treating, C6 cell is considerably less than matched group through the cell number of Recombinant Artificial basement membrane, and invasive ability is suppressed, difference have statistical significance (P< 0.05).Wherein,B3WithB4(relative invasion and attack rate is respectively 40.78% ± 3.72% and 20.36% ± 5.84%) is significantly stronger than crude drug berberine (relative mobility is 56.17% ± 4.00%) to the inhibitory action of invasion and attack.
Data above all applies SPSS 13.0 statistics software analysis.Mean testing uses variance analysis,P＜ 0.05 is that difference is statistically significant.
Strong migration with invasive ability is the key factor that restriction glioblastoma patient obtains long life cycle.But, current a large amount of cytotoxic drugs not only can not suppress migration and the invasion and attack of glioma effectively, its clinical practice of contrary drug resistance, bone marrow depression effect limits.Inventor finds the berberine lipophilic derivatives that the present invention provides, and can strengthen it to the propagation of C6 glioma cell, the inhibitory action that migrates and attack the while of increasing berberine lipophilic.
3. the Mitochondrially targeted property of berberinc derivate
Take the logarithm C6 and the U87 cell of trophophase, with 4 × 105Individual/ware is seeded in culture dish at the bottom of the special glass of laser confocal microscope cultivation 24h.Remove culture medium, be separately added into the berberine containing 1 μM, 9-O-dodecyl berberine or the culture fluid of 9-O-cetyl berberine, hatch 24h.Sucking supernatant, PBS (pH 7.4) washs three times, washs three times with Mitotracker Green FM (500nM) dyeing 30min, ice-cold PBS, observes and take pictures under laser confocal microscope.
Observe under laser co-focusing, in C6 (Fig. 6 A) and U87 (Fig. 6 B) glioma cell Berberine and lipophilic derivatives energy selective aggregation thereof in mitochondrion.The mitochondrion of C6 and U87 glioma cell carries out dyeing location (in green) by mitotracker green, and carries out laser co-focusing observation after berberine and derivant (in yellow) thereof hatch 24 hours.Display in all stacking charts (combined), yellow and green have good subcellular fraction to position altogether, are shown as yellow green after superposition.
In C6 cell, berberine is more evenly distributed in whole cell (including Cytoplasm and nucleus), and fluorescence intensity is more weak, it is shown that more weak Mitochondrially targeted property.Comparatively, berberinc derivate B3 and B4 fluorescence distribution substantially completely overlap with mitotracker green fluorescence, substantially there is no fluorescence at nuclear fractions, it is shown that the Mitochondrially targeted property higher compared with berberine.In U87 cell, B3 and B4 navigates in cell mitochondrial the most exactly, and fluorescence intensity is relatively strong, shows that it has been distributed to mitochondrion in large quantities, it is shown that than Mitochondrially targeted property higher in C6 cell.
Claims (5)
1. the compound or its salt of formula (I) application in preparation treatment glioblastoma tumor medicine:
Formula (I),
Wherein, R is C12-C16Alkyl.
Application the most according to claim 1, wherein, R is dodecyl or n-hexadecyl.
Application the most according to claim 1 and 2, wherein said salt is selected from hydrobromate, hydriodate, hydrofluoride, hydrochlorate, sulfate, nitrate, phosphate, citrate, acetate and lactate.
Application the most according to claim 1, wherein, described pharmaceutical pack is extra to the treatment effective composition of glioblastoma containing at least one.
Application the most according to claim 1, wherein, described pharmaceutical pack contains pharmaceutical excipient.
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