CN105079004A - Application of salpichrolide R in preparing medicines for treating adenoid cystic carcinoma of salivary gland - Google Patents
Application of salpichrolide R in preparing medicines for treating adenoid cystic carcinoma of salivary gland Download PDFInfo
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- CN105079004A CN105079004A CN201510590358.9A CN201510590358A CN105079004A CN 105079004 A CN105079004 A CN 105079004A CN 201510590358 A CN201510590358 A CN 201510590358A CN 105079004 A CN105079004 A CN 105079004A
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Abstract
The invention discloses application of salpichrolide R in preparing medicines for treating adenoid cystic carcinoma of salivary gland, and belongs to the field of medicine. Researches discover that the salpichrolide R has an effect of inducing SACC-M apoptosis, and an apoptosis rate is on the rise with the extension of drug action time and the increase in drug concentration; and the salpichrolide R can be further researched and developed as medicines for treating adenoid cystic carcinoma of salivary gland.
Description
Technical field
The present invention relates to the novelty teabag of compound S alpichrolideR, be specifically related to the application of SalpichrolideR in preparation treatment adenoid cystocarcinoma of salivary gland medicine.
Background technology
The people such as VivianaE.Nicotra separation and purification first goes out compound S alpichrolideR, and achievement is published in famous natural product magazine (WithanolideswithPhytotoxicActivityfromTwoSpeciesoftheGen usSalpichroa:S.origanifoliaandS.tristisvar.lehmannii, J.Nat.Prod., 2013,76,2219-2225).
Not yet there is this compound at present about the active reporter for the treatment of adenoid cystocarcinoma of salivary gland.
Summary of the invention
The object of the present invention is to provide the medical usage of a kind of SalpichrolideR.
Above-mentioned purpose is achieved by the following technical solution:
The application of SalpichrolideR in preparation treatment adenoid cystocarcinoma of salivary gland medicine, described SalpichrolideR chemical structural formula is as follows,
Further, described adenoid cystocarcinoma of salivary gland is adenoid cystocarcinoma of salivary gland SACC-M.
Detailed description of the invention
Essentiality content of the present invention is further illustrated below in conjunction with embodiment.
The separation preparation of embodiment 1:SalpichrolideR and structural identification
The preparation method of SalpichrolideR is with preparation method (the WithanolideswithPhytotoxicActivityfromTwoSpeciesoftheGen usSalpichroa:S.origanifoliaandS.tristisvar.lehmannii of bibliographical information, J.Nat.Prod., 2013,76,2219-2225).
Structural identification: white amorphous powder; Comprehensive HR-ESI-MS, the molecular formula of this compound is C
28h
38o
6, degree of unsaturation is 10.Hydrogen nuclear magnetic resonance modal data δ
h(ppm, DMSO-d
6, 400MHz): H-2 (5.91, dd, J=10.0, 2.2Hz), H-3 (6.67, ddd, J=10.0, 5.0, 2.2Hz), H-4 (1.75, dd, J=19.6, 5.0Hz), H-4 (3.04, dt, J=19.6, 2.8Hz), H-6 (3.03, d, J=5.1Hz), H-7 (1.52, m), H-7 (2.22, dt, J=14.8, 5.5Hz), H-8 (1.68, m), H-9 (1.73, m), H-11 (2.38, m), H-11 (1.37, m), H-12 (1.67, m), H-12 (1.42, m), H-14 (1.13, dd, J=10.3, 4.3Hz), H-15 (4.35, dd, J=4.8, 2.8Hz), H-16 (5.57, d, J=2.6Hz), H-18 (1.00, s), H-19 (1.38, s), H-20 (2.11, m), H-21 (0.92, d, J=6.9Hz), H-22 (3.69, ddd, J=10.5, 7.5, 2.3Hz), H-23 (1.53, m), H-23 (1.95, dd, J=14.3, 2.5Hz), H-26 (4.94, s), H-27 (1.40, s), H-28 (1.37, s), carbon-13 nmr spectra data δ
c(ppm, DMSO-d
6, 50MHz): 202.2 (C, 1-C), 128.8 (CH, 2-C), 142.0 (CH, 3-C), 33.9 (CH
2, 4-C), 64.7 (C, 5-C), 58.2 (CH, 6-C), 27.4 (CH
2, 7-C), 26.1 (CH, 8-C), 38.7 (CH, 9-C), 48.6 (C, 10-C), 21.6 (CH
2, 11-C), 34.0 (CH
2, 12-C), 46.5 (C, 13-C), 59.1 (CH, 14-C), 73.2 (CH, 15-C), 123.9 (CH, 16-C), 162.3 (C, 17-C), 22.2 (CH
3, 18-C), 15.1 (CH
3, 19-C), 35.8 (CH, 20-C), 17.0 (CH
3, 21-C), 66.2 (CH, 22-C), 34.4 (CH
2, 23-C), 64.3 (C, 24-C), 63.5 (C, 25-C), 91.1 (CH, 26-C), 16.2 (CH
3, 27-C), 18.5 (CH
3, C-28).Structural identification data are consistent with bibliographical information, therefore can determine that compound prepared by the present invention is the SalpichrolideR of bibliographical information.
The pharmacological action test of embodiment 2:SalpichrolideR
One, material and instrument
Salivary Adenoid Cystic Carcinoma Cell system SACC-M is purchased from Medical College, Shanghai Communication Univ.'s Oral and Maxillofacial Surgery laboratory.SalpichrolideR makes by oneself, and HPLC normalization purity is greater than 98%.RPMI-1640 culture medium, trypsin are purchased from SIGMA company of the U.S..Standard hyclone is purchased from Xingtai City Huifeng biotechnology research institute.Annexin-v-FITC/PI test kit is purchased from Beijing Bao Sai Bioisystech Co., Ltd.PBS is purchased from Tianjin recovery fine chemistry industry institute.MTT powder is purchased from Amresco company.Na2EDTA (EDTA) is purchased from Beijing Yili Fine Chemicals Co., Ltd..Penicillin, streptomycin are purchased from North China pharmaceutical Co. Ltd.Dimethyl sulfoxide (DMSO) is purchased from Tianjin Standard Science company limited.Giemsa dye liquor is purchased from Zhuhai Bei Suo Technology Co., Ltd..
XYJ type medical treatment clean work station (Purifying Equipment Co., Ltd., Suzhou), BB5060/BB16 CO2 gas incubator (Shanghai Heraeus company), CX21 optical microscope (Japanese OLYMPUS), XD-10198101 inverted microscope (south of the River company), flow cytometer (BectionDickinsonFacscaLibur), the full-automatic microplate reader of WeLLscanMK3 (Finland LabsystemsDragon), BFX5-320 type low speed centrifuge (Chinese Shanghai Lu Nan scientific instrument related factory), GF-300 type electronic balance (JapanA & DCompany, Limited), 725 type ultra cold storage freezers (FormaScientific.Inc), micro sample adding appliance (EPPENDORF company).
One, test method
1, cell culture
1.1 cell recovery
Prepare 37 DEG C of constant water bath box, taken out by the SACC-M cell cryopreservation tube of Liquid nitrogen storage, directly drop into water bath, shake gently, after it melts rapidly, from water tank, take out cryopreservation tube, this step should be fast, should complete within 30-60 second.Afterwards to open cryopreservation tube after the 75% alcohol wipe sterilization mouth of pipe, by the sucking-off of pipe inner cell suspension, instillation 50mL centrifuge tube also drips more than the 10 times RPMI-1640 culture fluid containing 10% serum in centrifuge tube, blow and beat gently, become cell suspension, low-speed centrifugal (1000 revs/min) is after 5 minutes, suck supernatant, wash once with the RPMI-1640 culture fluid of 10% again, and recentrifuge, after sucking supernatant, with the RPMI-1640 culture fluid containing 10% hyclone, cell dilution is become cell suspension, be inoculated in 50mL culture bottle, unscrew bottle cap gently, then CO is put into
2cultivate in incubator, second day changes a culture fluid, changes culture fluid afterwards after culture fluid color is by light red flavescence, when observation of cell climbs 80% of full bottle diapire, carries out passage.
1.2 passage
First after using elbow straw to draw original fluid, repeatedly blow and beat at the bottom of bottle, remove old culture fluid in bottle afterwards, mixture slaking liquid (0.25% trypsin and 0.04%EDTA1:1) a small amount of (being advisable covering completely at the bottom of culture bottle) is added in bottle, at the bottom of slow shake body makes Digestive system soak bottle, then suck most of Digestive system, only stay and digest on a small quantity.Culture bottle is placed in CO
2in incubator, (or under 25 DEG C of room temperatures) digest, and take out culture bottle, fell and put basis of microscopic observation after 2-5 minute, when discovery kytoplasm retraction, intercellular substance increase, part cell becomes circle from polygon, and when existing cell starts to suspend, should stop digestion immediately.The Digestive system that sucking-off is remaining, adds the appropriate culture fluid not containing serum, rotates culture bottle gently, after washing out residual Digestive system, by its sucking-off, then add new culture fluid, repeatedly blows and beats at the bottom of bottle, make subsides with elbow straw after drawing culture in glassware liquid.Cell detachment at the bottom of bottle, forms cell suspension, and when noting blowing and beating, action wants soft, in case overexert, make cell damage.Drawing cell suspension is placed in centrifuge tube, after centrifugal, removes supernatant, collecting cell, and add the appropriate RPMI-1640 culture fluid containing 10% hyclone, cell is blown and beaten into suspension, counting, with 1.0 × 10
5/ mL concentration is seeded in new culture bottle.
2, MTT colorimetric test
(1) inoculating cell: trophophase cell of taking the logarithm, making concentration with the RPMI-1640 containing 10% hyclone is 2 × 10
5the single cell suspension of/mL, is inoculated in 96 orifice plates, every hole 100 μ L.(2) cultured cell: above-mentioned 96 orifice plates are placed in CO
2incubator, at 37 DEG C, 5%CO
2and under saturated humidity condition, continue to cultivate 24h.(3) dosing: the every hole of medication group adds the SalpichrolideR medicinal liquid 100 μ L after dilution, make its final concentration be 2.5,5.0,7.5,10,12.5mmol/L; The every hole of matched group adds 100 μ L not containing the RPMI-1640 of serum.Medication group and matched group are often organized and are all established 6 multiple holes, and establish zeroing hole, continue to cultivate after dosing.(4) colour generation: after continuing to cultivate 24h, 48h and 72h, medication group and the every hole of matched group add the MTT solution 20 μ L that concentration is 5g/L, after continuing to cultivate 4h, suck whole supernatant, the dimethyl sulfoxide (comprising zeroing hole) of 200 μ L is added in each hole, put vibration on agitator and shake up 1 minute, crystallization is fully dissolved.(5) colorimetric: be placed in microplate reader by 96 orifice plates is 490nm place at wavelength, measures each hole absorbance (A).Repeat experiment 3 times, get its meansigma methods.(6) calculate and draw: cell proliferation inhibition rate=(1-medication group average A-value/matched group average A-value) × 100%.According to the growth inhibition ratio of each concentration of above formulae discovery, be then transverse axis with time, suppression ratio is the longitudinal axis, draws the suppression ratio curve of 5 concentration.
3, Flow cytometry
The process of 3.1 cells
After cell inoculation, cultivation, trophophase cell of taking the logarithm, with 2 × 10
5/ mL concentration is inoculated in 6 orifice plates, then dosing, if time contrast groups, concentrations versus's group and matched group (normal apoptotic group): (1) time contrast groups: cell is inoculated in 6 orifice plates, cultivate 24 hours, after cell attachment, with suction pipe along each hole wall by thorough for original fluid sucking-off, the final concentration then adding equivalent in each hole is the culture fluid (medication group) that 12.5mmol/L contains SalpichrolideR medicinal liquid.Add not containing SalpichrolideR culture fluid as a control group, respectively continue cultivate 24h, 48h, 72h.(2) concentrations versus's group: cell inoculation also, after adherent success, thoroughly to exhaust original fluid with suction pipe, add respectively in each hole final concentration be 2.5,5.0,7.5,10,12.5mmol/L containing SalpichrolideR culture fluid (medication group).Matched group adds not containing the culture fluid of SalpichrolideR, continues to cultivate 72h.(3) normal apoptotic group: inoculating cell also, after adherent success, removes original fluid in each hole, adds not containing the culture fluid of SalpichrolideR, continues to cultivate 24h, 48h, 72h.
The preparation of 3.2 samples
(1) collecting cell: draw 6 orifice plate each hole supernatant respectively, add in centrifuge tube, after getting each diapire of PBS liquid 2mL rinsing 6 orifice plate, pour in same centrifuge tube, then 2mL mixture slaking drop is entered peptic cell in each hole, slow wave and culture plate makes Digestive system soak its diapire completely, after digestion 2min, Digestive system is sucked each centrifuge tube, add 2mL culture fluid again and blow and beat each hole diapire, after cell thoroughly takes off wall, suck in centrifuge tube.(2) centrifuge cell: centrifuge tube is placed in low speed centrifuge, after the centrifugal 5min of 1500rpm, abandoning supernatant; Add 12mLPBS liquid re-suspended cell, then in the centrifugal 5min of 1500rpm, abandon supernatant; Cell suspension after re-suspended cell, is drawn in the special upper machine pipe of flow cytometer, after the centrifugal 5min of 1500rpm, abandons supernatant by the PBS liquid instilling 3mL again in centrifuge tube.
3.3Annexin-V-FITC and PI double labelling living cells method detects apoptosis
(1) in streaming pipe, the Annexin-V-FITC of 10 μ L and the PI (two kinds of reagent do not mix mutually, do not contact with pipe floor cells) of 5 μ L is added with micro sample adding appliance.(2) add 200 μ L binding buffer liquid, Annexin-V-FITC with PI punching is mixed mutually with cell at the bottom of pipe, and blows and beats gently, form single cell suspension.(3) lucifuge is reacted 15min or is put into 4 DEG C of refrigerator 30min.(4) add 300 μ LPBS binding buffer liquid before upper machine, and blow and beat gently, then go up machine (flow cytometer) at once and detect.Light source is 488nm argon ion laser, and 10000, every part of specimen collection cell, fluoresced green after FITC is stimulated, PI sends out red fluorescence.(5) through flow cytometry analysis, obtain the scatterplot be made up of four quadrants, on figure, each point represents a cell, all has two parameter values.Upper left Q1 quadrant represents the damaging cells (An-PI+) occurred in cell harvesting process, upper right Q2 quadrant represents non-viable apoptotic cell and non-viable non-apoptotic cell (An+PI+), lower-left Q3 quadrant represents normal cell (An-PI-), and bottom right Q4 quadrant represents viable apoptotic cell (An+PI-).
4, statistical analysis
Using statistics software SPSS10.0 carries out statistical analysis.(1) use the variance analysis of Factorial Design, the growth inhibition ratio of SalpichrolideR to SACC-M cell between same medicine concentration different disposal time, each group of same treatment time different pharmaceutical concentration is compared between two.(2) use simple correlation analysis, analyze growth inhibition ratio and the dependency between action time, between suppression ratio and drug level, draw correlation coefficient r, and t inspection is carried out to r value, thus draw drug effect in time with the variation relation of concentration.(3) each medication group using the u-test flow cytometric art of binomial distribution to detect and the apoptosis rate of matched group carry out statistical analysis.
Two, result and conclusion
1, MTT colorimetric test
(1) experiment shows that SalpichrolideR has obvious inhibited proliferation to SACC-M cell, drug effect 24,48, the highest suppression ratio can reach 80.4% (table 1) after 72h.(2) suppression ratio between same treatment time, variable concentrations group compares notable difference (P<0.01); Same concentrations group, suppression ratio between the different disposal time have notable difference (P<0.01); Drug level and processing time non-interaction action (P=0.901).(3) compare between two between group: between same time, variable concentrations group, suppression ratio has notable difference (P<0.01); After process 24h, 48h, 72h, between same concentration, different time group, there is notable difference (P<0.01).(3) drug effect (suppression ratio) all has positive correlation with action time and drug level, and suppression ratio and action time have positive correlation (r=0.291, P=0.042); Suppression ratio and concentration have positive correlation (r=0.926, P<0.001).
2, Flow cytometry
The apoptosis situation of 2.1 cellular control units
Matched group (normal apoptotic group) cell all belongs to natural apoptosis, apoptosis rate when its apoptosis rate is 4.9%, 48h during 24h is the apoptosis rate of 7.1%, 72h is 6.1% (table 2), apoptosis rate is little with incubation time difference, not statistically significant.
2.2 medication group cells change apoptosis situation in time
After 12.5mmol/LSalpichrolideR process 24h, its apoptosis rate is 25.5%; After process 48h, apoptosis rate rises to 40.9%, mostly is early apoptosis; After process 72h, apoptosis rate is 67.6%, mostly is late apoptic, and apoptosis rate extends along with the processing time and obviously rises (table 2), and between each time group, apoptosis rate has statistical significance (P<0.01).
2.3 medication group cells are with concentration change apoptosis situation
After process 72h, 2.5,5.0,7.5,10.0, the apoptosis rate of 12.5mmol/LSalpichrolideR group is respectively 16%, 24.2%, 26.1%, 66.3%, 67.6%, and when drug level reaches 12.5mmol/L, late apoptic rate obviously increases (table 2), and between each drug level group, apoptosis rate has statistical significance (P<0.01).
Conclusion, this research shows that SalpichrolideR has the effect of induction SACC-M apoptosis, and apoptosis rate with the prolongation of drug treating time and the increase of Drug level in rising trend.Application SalpichrolideR treats adenoid cystic carcinoma likely becomes the very promising Therapeutic Method of one.
The different suppression ratio action time (mean value ± SD) of table 1 variable concentrations
Table 2SalpichrolideR induces SACC-M apoptosis situation
| Concentration-time | Normal cell (%) | Early apoptosis (%) | End-stage apoptotic (%) | Damaging cells (%) |
| 0mmol/L—24h | 93.2 | 0.5 | 4.9 | 1.4 |
| 0mmol/L—48h | 90.4 | 2.3 | 7.1 | 0.3 |
| 0mmol/L—72h | 87.5 | 6.1 | 6.1 | 0.2 |
| 12.5mmol/L—24h | 74.4 | 11.8 | 13.7 | 0.1 |
| 12.5mmol/L—48h | 59 | 35.4 | 5.5 | 0 |
| 12.5mmol/L—72h | 32.4 | 21.3 | 46.3 | 0.1 |
| 2.5mmol/L—72h | 82.4 | 1.5 | 14.5 | 1.7 |
| 5.0mmol/L—72h | 75.7 | 19.3 | 4.9 | 0.1 |
| 7.5mmol/L—72h | 73.9 | 15.2 | 10.9 | 0 |
| 10.0mmol/L—72h | 33.6 | 58.7 | 7.6 | 0.1 |
Claims (2)
- The application of 1.SalpichrolideR in preparation treatment adenoid cystocarcinoma of salivary gland medicine, described SalpichrolideR chemical structural formula is as follows,
- 2. the application of SalpichrolideR according to claim 1 in preparation treatment adenoid cystocarcinoma of salivary gland medicine, is characterized in that: described adenoid cystocarcinoma of salivary gland is adenoid cystocarcinoma of salivary gland SACC-M.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105294817A (en) * | 2015-11-25 | 2016-02-03 | 吕涛 | Novel triterpenoid, preparation method and medical application thereof |
| CN105541961A (en) * | 2016-03-04 | 2016-05-04 | 宋晓梅 | Withanolide compounds for treating adenoid cystic carcinoma of salivary gland |
-
2015
- 2015-09-16 CN CN201510590358.9A patent/CN105079004A/en not_active Withdrawn
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105294817A (en) * | 2015-11-25 | 2016-02-03 | 吕涛 | Novel triterpenoid, preparation method and medical application thereof |
| CN105541961A (en) * | 2016-03-04 | 2016-05-04 | 宋晓梅 | Withanolide compounds for treating adenoid cystic carcinoma of salivary gland |
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Application publication date: 20151125 |