CN105147647A - Application of d-borneol serving as antitumor drug sensitizer - Google Patents
Application of d-borneol serving as antitumor drug sensitizer Download PDFInfo
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- CN105147647A CN105147647A CN201510511663.4A CN201510511663A CN105147647A CN 105147647 A CN105147647 A CN 105147647A CN 201510511663 A CN201510511663 A CN 201510511663A CN 105147647 A CN105147647 A CN 105147647A
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A61K31/00—Medicinal preparations containing organic active ingredients
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
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Abstract
The invention discloses an application of d-borneol serving as an antitumor drug sensitizer. According to the application of d-borneol serving as the antitumor drug sensitizer, d-borneol serving as the antitumor drug sensitizer is used for assisting in antitumor drugs. The inventor discovers that after the concentration of d-borneol which has no remarkable growth influence on cells is increased, the sensitivity of non-resistant cells to antitumor chemical drugs is greatly improved, that is, the concentration of the antitumor chemical drugs for killing tumor cells can be reduced on the basis of addition of d-borneol, the curative effect is not reduced, toxic and side effects of the chemical drugs are reduced along with reduction of the concentration of the chemical drugs, and normal cells of people are protected. D-borneol has low toxicity for normal cells of people and does not have remarkable influence basically, and accordingly, d-borneol is a potential high-efficient low-toxicity chemical drug sensitizer.
Description
Technical field
The present invention relates to the purposes of dextro Borneolum Syntheticum, particularly dextro Borneolum Syntheticum is as the application of antitumor drug sensitizer.
Background technology
At present, the treatment of cancer is mainly based on excision, and because cancerous cell easily shifts, therefore operation can not be effected a radical cure, and is usually aided with radiation and chemotherapy.Although radiotherapy is faster than Chemotherapy effect, also bring a lot of side effect to patient, therefore, chemotherapy (chemotherapy) is posted to be to alleviate the Main Means of patient's misery.
Since half a century, chemotherapy of tumors has had and has developed rapidly.Various antitumor drug, if the sequential use such as amycin, daunorubicin, cisplatin, 5-fluorouracil, paclitaxel are in clinical, brings Gospel to patient.But, chemotherapy also inevitably produces many toxic and side effects, as the antitumor drug such as amycin and daunorubicin be applied to human body after can bring certain toxic and side effects to heart, can cause that Sudden cardiac is overrun, acute heart failure, dyspnea etc.Can cause allergic reaction after paclitaxel application human body, occur the symptom such as hypotension, urticaria, respiratory distress.Therefore, the antitumous effect how farthest playing medicine can reduce again the hot issue that its toxic and side effects is research all the time.For this reason, seek a class and can not only improve the sensitivity of tumor cell to antitumor drug, and the chemicals sensitizer that can significantly improve antitumor drug tumor killing effect in the treatment just to seem particularly important.
Dextro Borneolum Syntheticum a kind ofly from the cloudy fragrant chemical type borneol tree branches and leaves of Lauraceae, extracts the native compound obtained, and its chemical structural formula is as follows:
As everyone knows, dextro Borneolum Syntheticum swells and ache for excess syndrome of stroke coma, conjunctival congestion and swelling pain, sore throat aphtha, skin infection, does not hold back and angina pectoris etc. after bursting.At present, not yet relevant dextro Borneolum Syntheticum has the report of antitumor drug sensitizer function.
Summary of the invention
Primary and foremost purpose of the present invention is that the shortcoming overcoming prior art is with not enough, provides dextro Borneolum Syntheticum as the application of antitumor drug sensitizer.
Another object of the present invention is to provide a kind of antitumor drug sensitizer.
Another object of the present invention is to provide a kind of antitumor drug.
Object of the present invention is achieved through the following technical solutions: dextro Borneolum Syntheticum, as the application of antitumor drug sensitizer, is applied as antitumor drug sensitizer assistant anti-tumor drug by dextro Borneolum Syntheticum.
Described dextro Borneolum Syntheticum is natural d-borneol.
Described antitumor drug is preferably anti-tumor medicine thing.
Described anti-tumor medicine thing comprises: daunorubicin, amycin, darubicin, epirubicin, paclitaxel, lentinan, vincaleucoblastine, vincristine, tamoxifen, Formestane, Anastrozole, flutamide, 5-fluorouracil, methotrexate, cisplatin, carboplatin, oxaliplatin, carmustine, toremifene, ftorafur, curcumin, demethoxycurcumin, bisdemethoxycurcumin and phosphinothioylidynetrisaziridine etc.; Be preferably the one in curcumin, demethoxycurcumin and bisdemethoxycurcumin or at least two kinds.
Described tumor mainly refers to non-drug-resistant tumor, as hepatocarcinoma, pulmonary carcinoma, malignant melanoma, breast carcinoma, colon cancer, rhinocarcinoma, bladder cancer, cervical cancer, gastric cancer, the esophageal carcinoma and carcinoma of prostate.
A kind of antitumor drug sensitizer prepares based on the application of dextro Borneolum Syntheticum as antitumor drug sensitizer, and its excipient allowed by dextro Borneolum Syntheticum and medicine or carrier form.
A kind of antitumor drug, is made up of tumour medicine sensitizer and antitumor drug, is namely made up of dextro Borneolum Syntheticum, excipient or carrier and antitumor drug.
Described antitumor drug is preferably anti-tumor medicine thing.
Described anti-tumor medicine thing comprises: daunorubicin, amycin, darubicin, epirubicin, paclitaxel, lentinan, vincaleucoblastine, vincristine, tamoxifen, Formestane, Anastrozole, flutamide, 5-fluorouracil, methotrexate, cisplatin, carboplatin, oxaliplatin, carmustine, toremifene, ftorafur, curcumin, demethoxycurcumin, bisdemethoxycurcumin and phosphinothioylidynetrisaziridine etc.; Be preferably the one in paclitaxel, curcumin, demethoxycurcumin and bisdemethoxycurcumin or at least two kinds.
Described tumor mainly refers to non-drug-resistant tumor, i.e. cancer, as hepatocarcinoma, pulmonary carcinoma, malignant melanoma, breast carcinoma, colon cancer, rhinocarcinoma, bladder cancer, cervical cancer, gastric cancer, the esophageal carcinoma and carcinoma of prostate.
Described antitumor drug is preferably made up of dextro Borneolum Syntheticum, excipient or carrier and curcumin; It can be used for treating melanoma.
Described curcumin and described dextro Borneolum Syntheticum 73674 ~ 294696:400000 proportioning in mass ratio; Preferred 73674:400000 proportioning in mass ratio.
Described antitumor drug is preferably made up of dextro Borneolum Syntheticum, excipient or carrier and curcumin chemical compounds; It can be used for Hepatoma therapy.
Described curcumin chemical compounds is at least one in curcumin, demethoxycurcumin and bisdemethoxycurcumin.
Described dextro Borneolum Syntheticum and described curcumin chemical compounds are by quality mol ratio 2 ~ 8 μ g:2 ~ 8nmol proportioning.
Preferably, described dextro Borneolum Syntheticum and described curcumin are by quality mol ratio 1 μ g:1nmol proportioning; Described dextro Borneolum Syntheticum and described demethoxycurcumin are by quality mol ratio 1 μ g:2nmol proportioning; Described dextro Borneolum Syntheticum and described bisdemethoxycurcumin are by quality mol ratio 1 μ g:2nmol proportioning.
The present invention has following advantage and effect relative to prior art:
(1) present invention finds the new opplication of dextro Borneolum Syntheticum as anti-tumor medicine thing sensitizer.
(2) add used to cell without the dextro Borneolum Syntheticum concentration of obvious growth effect after, the sensitivity of non-mdr cell antagonism tumor chemicals increases greatly, namely adding on the basis with dextro Borneolum Syntheticum, the concentration being used for the anti-tumor medicine thing of killing tumor cell can be reduced, but curative effect does not reduce.And the toxic and side effects of chemicals reduces along with the reduction of its concentration, protection human normal cell.
(3) dextro Borneolum Syntheticum is low to human normal cell's toxicity, substantially has no significant effect, and is a kind of chemicals sensitizer of potential high-efficiency low-toxicity.
Accompanying drawing explanation
Fig. 1 is that Malignant Melanoma A 375 Cells is respectively through the testing result figure after curcumin and curcumin+dextro Borneolum Syntheticum process.
Fig. 2 is that breast cancer cell MCF-7 cell is respectively through the testing result figure after curcumin and curcumin+dextro Borneolum Syntheticum process.
Fig. 3 is that different disposal group affects result figure to curcumin content in cell;
Wherein: matched group represents and do not add NB or Cur process, NB group represents interpolation 40 μ g/mlNB process, and Cur group represents interpolation 20 μMs of Cur process, and interpolation 20 μMs of Cur process after 40 μ g/mlNB process 12h are first added in the expression of NB+Cur group.
Fig. 4 is that different disposal group lures a result of variations figure leading SubG-1 content in A375 cell;
Wherein, "-" expression is not added, and "+" represents interpolation.
Fig. 5 is the testing result figure that different disposal group activates Caspase3/8/9 in A375 cell;
Wherein, matched group represents and does not add NB or Cur process, and NB group represents interpolation 40 μ g/mlNB process, and Cur group represents interpolation 20 μMs of Cur process, and interpolation 20 μMs of Cur process after 40 μ g/mlNB process 12h are first added in the expression of NB+Cur group.
Fig. 6 is the testing result figure of ROS content in different disposal group induction A375 cell;
Wherein, matched group represents and does not add NB or Cur process, and NB group represents interpolation 40 μ g/mlNB process, and Cur group represents interpolation 20 μMs of Cur process, and interpolation 20 μMs of Cur process after 40 μ g/mlNB process 12h are first added in the expression of NB+Cur group.
Fig. 7 is that the Different treatments of dextro Borneolum Syntheticum and curcumin affects result figure to HepG2 cell survival rate; Different letter representation statistically has significant difference (P<0.05), and same letter represents statistically do not have significant difference.
Fig. 8 is that the Different treatments of dextro Borneolum Syntheticum and curcumin affects result figure to HepG2 cell cycle.
What the Different treatments of Fig. 9 dextro Borneolum Syntheticum and curcumin changed ROS in HepG2 cell affects result figure.
Figure 10 is that the Different treatments of dextro Borneolum Syntheticum and demethoxycurcumin affects result figure to HepG2 cell survival rate; Different letter representation statistically has significant difference (P<0.05), and same letter represents statistically do not have significant difference.
Figure 11 is that the Different treatments of dextro Borneolum Syntheticum and demethoxycurcumin affects result figure to HepG2 cell cycle.
Figure 12 be the Different treatments of dextro Borneolum Syntheticum and demethoxycurcumin ROS in HepG2 cell is changed affect result figure.
Figure 13 is that the Different treatments of dextro Borneolum Syntheticum and bisdemethoxycurcumin affects result figure to HepG2 cell survival rate; Different letter representation statistically has significant difference (P<0.05), and same letter represents statistically do not have significant difference.
Figure 14 is that the Different treatments of dextro Borneolum Syntheticum and bisdemethoxycurcumin affects result figure to HepG2 cell cycle.
Figure 15 be the Different treatments of dextro Borneolum Syntheticum and bisdemethoxycurcumin ROS in HepG2 cell is changed affect result figure.
Detailed description of the invention
Below in conjunction with embodiment and accompanying drawing, the present invention is described in further detail, but embodiments of the present invention are not limited thereto.
Embodiment 1 dextro Borneolum Syntheticum increases the sensitivity of non-cells of resistant tumors to chemicals
(1) experiment material
Human melanoma cell line A375 used in this experiment is purchased from American Type Culture collection warehousing (ATCC, Manassas, VA); Dextro Borneolum Syntheticum, Chinese medicine and biological products assay institute; Curcumin, is purchased from Sigma company; Methyl thiazoly tetrazolium assay (MTT) powder and dimethyl sulfoxide (DMSO), Sigma Co., USA; DMEM culture fluid and pancreatin, American I nvitrogen (Carlsbad, CA) company; 96 orifice plates, Corning company of the U.S..
(2) experimental technique
By A375 cell culture in the DMEM culture fluid containing percent by volume 10% hyclone, cell culture is after 3 days, and the growth conditions of observation of cell and cell density, when cell grows to 80%, carry out passage.By pancreatin (routine operation, working solution concentration is 0.25%, lower with) cell after digestion is lower is transferred in centrifuge tube, supernatant discarded after the centrifugal 3min of 1000rpm, add the DMEM culture medium 3ml containing 10% (v/v) hyclone, rifle is inhaled and is made a call to more than 10 times, and cell must be made evenly resuspended, and adjusting cell density with fresh culture after getting 10 μ l cell suspension Trypan Blue meter viable counts is 1.8 × 10
4cells/ml is inoculated in 96 well culture plates, every hole 100 μ l; After 24h, one group of chemicals 100 μ l adding variable concentrations, another group adds 50 μ l of 40 μ g/mL dextro Borneolum Syntheticum 50 μ l and variable concentrations chemicals.After 72h, the growth conditions of observation of cell of taking pictures under the microscope.Then every hole adds MTT (5mg/ml) 20 μ l, puts into incubator and continues to cultivate 4h.The supernatant in hole is carefully sucked after 4h.Every hole adds the DMSO of 150 μ l afterwards, and concussion 10min, 490nm survey light absorption value.Calculate cell survival rate, cell survival rate=(OD
experiment-OD
blank)/(OD
contrast-OD
blank) × 100%.After calculating, different disposal group and cell survival rate are done block diagram, and be t-inspection carry out significant difference analysis design mothod result.With origin8.0 computed in software half suppression ratio (IC
50), and the IC to tumor cell after pressing formulae discovery dose degradation index (DoseReductionIndex, DRI)=certain chemicals alone
50/ add the IC50 to tumor cell after dextro Borneolum Syntheticum with chemicals.Often test in triplicate.
(3) experimental result
This experiment adopt dextro Borneolum Syntheticum concentration to A375 without obvious growth inhibited.Through preliminary experiment, the concentration determining dextro Borneolum Syntheticum is 40 μ g/ml.As shown in Figure 1, use after concentration is the dextro Borneolum Syntheticum of 40 μ g/ml adding, chemicals curcumin (Curcumin, Cur) sensitivity of the A375 malignant melanoma cell of this experiment is improved greatly, its Susceptible change index-dose degradation index (DRI) is 1.72 (see table 1), illustrates that effect of enhanced sensitivity is obvious.
Table 1 adds with the change of curcumin after dextro Borneolum Syntheticum to IC50 and DRI of A375 cell
Embodiment 2 dextro Borneolum Syntheticum increases the sensitivity of non-cells of resistant tumors to chemicals
(1) experiment material
Breast cancer cell MCF-7 used in this experiment is purchased from American Type Culture collection warehousing (ATCC, Manassas, VA); Dextro Borneolum Syntheticum, Chinese medicine and biological products assay institute; Curcumin, is purchased from Sigma company; Methyl thiazoly tetrazolium assay (MTT) powder and dimethyl sulfoxide (DMSO), Sigma Co., USA; DMEM culture fluid and pancreatin, American I nvitrogen (Carlsbad, CA) company; 96 orifice plates, Corning company of the U.S..
(2) experimental technique
Operation is with embodiment 1.
(3) experimental result
This experiment adopt dextro Borneolum Syntheticum concentration to MCF-7 without obvious growth inhibited.Through preliminary experiment, the concentration determining dextro Borneolum Syntheticum is 40 μ g/ml.As shown in Figure 2, use after concentration is the dextro Borneolum Syntheticum of 40 μ g/ml adding, the sensitivity of chemicals curcumin (Curcumin) to the MCF-7 breast cancer cell of this experiment improves greatly, its Susceptible change index-dose degradation index (DRI) is 3.86 (see table 1), illustrates that effect of enhanced sensitivity is obvious.
Table 2 adds with the change of curcumin after dextro Borneolum Syntheticum to IC50 and DRI of MCF-7 cell
Embodiment 340 μ g/mlNB+20 μM of Cur effect A375 cell
1, the content detection of curcumin in A375 cell
(1) experiment material
Containing the NaOH solution (concentration of NaOH is 0.1mol/L) of 1% (v/v) TritionX-100, be called for short cell pyrolysis liquid.
(2) experimental technique
Take the logarithm the A375 cell of trophophase, through trypsinization, counting, with 8 × 10
4individual cells/well is inoculated in 96 well culture plates, be placed in incubator (37 DEG C, 5%CO
2) cultivate after 24h.Add after 40 μ g/mlNB, 20 μMs of Cur and 40 μ g/mlNB+20 μM Cur (this group is for first adding Cur again with after NB process 12h) process 0.5h, 1h, 2h and 4h respectively, pump culture medium, add 1mLPBS (0.01M, pH7.2, down together) cleaning cell removes extracellular dextro Borneolum Syntheticum and curcumin for 3 times, then 200 μ L cell pyrolysis liquids are added, after vibration 10min, under fluorescence microplate reader, carry out the mensuration of curcumin content.Wherein, excitation wavelength is 425nm, and emission wavelength is 545nm.Carry out repeating experiment for three times.
(3) experimental result and analysis
As shown in Figure 3, contrast blank group, in NB group process its cell rear, curcumin content is without significant change, but NB+Cur group but significantly improves the content of Cur in cell and is time dependence.0.5,1,2, after 4h, in NB+Cur group, the content of Cur brings up to 3.23,3.47,3.99 and 4.36 (10 from 2.57 of matched group respectively
-9μ g), than 3.21,3.40,3.55 and 3.86 (10 of Cur group
-9μ g) improve 1.01,1.02,1.12 and 1.13 times respectively.Experimental result shows, dextro Borneolum Syntheticum improves curcumin significantly in intracellular absorption.
2, flow cytometry
(1) experiment material
PI dyestuff (concentration is 50 μ g/ml)
(2) experimental technique
Take the logarithm the A375 cell of trophophase, through trypsinization, counting, with 2 × 10
4individual cells/well is inoculated in 6 orifice plates, be placed in incubator (37 DEG C, 5%CO
2) cultivate after 24h.Add 40 μ g/mlNB respectively, 20 μMs of Cur, and after 40 μ g/mlNB+20 μM Cur (this group is for first adding Cur again with after NB process 12h) processes 72h, pump culture medium, add 1mLPBS cleaning cell and remove extracellular dextro Borneolum Syntheticum and curcumin 3 times, then trypsinization is added, the centrifugal 5min of 1500rpm, collecting cell, add 70% ethanol 1mL of-20 DEG C of pre-coolings, mixing, 4 DEG C are spent the night fixing, next day, the centrifugal 5min of 1500rpm, remove supernatant, 2 times are washed with PBS, add 500 μ lPI dye liquors, after after mixing, lucifuge leaves standstill 20min, cross 300 mesh sieves, upper machine testing.10000 cells at least analyzed by each sample.
(3) results and analysis
As shown in Figure 4, compare matched group, do not occur obvious Apoptotic cell peak in NB group, after its SubG-1 is only the process of 2.5%, Cur group, Apoptotic cell peak brings up to 27.5% from 1.4% of matched group, and compare Cur group, apoptosis peak in NB+Cur group brings up to 75% from 27.5%, and fluidic cell result shows, after adding NB, the subG-1 peak that can significantly improve in Cur inducing cell raises, and shows NB and Cur combined induction apoptosis.
3, Caspase (the aspartic acid proteolytic enzyme containing cysteine) Activity determination
(1) experiment material
Caspase substrate Caspase3, Caspase8, Caspase9
(2) experimental technique
Take the logarithm the cell of trophophase, through trypsinization, counting, with 10 × 10
4individual cells/well is inoculated in 10cm ware, be placed in incubator (37 DEG C, 5%CO
2) cultivate after 24h.Add 40 μ g/mlNB, 20 μMs of Cur and 40 μ g/mlNB+20 μM Cur process 72h respectively, then collecting cell, (RIPA lysate is by 50mMTris-HCl (pH7.4), 150mMNaCl to add a certain amount of RIPA cell pyrolysis liquid, 1%NonidetP-40 and 0.1%SDS is formulated, addition visual cell number and determine), after hatching 1h on ice, centrifugal 30min under 11000g, gets supernatant and transfers in new EP pipe.Then BCA kit measurement protein concentration is adopted.Then Caspase substrate (Caspase3,8,9) is pipetted respectively, add in 96 hole fluorescent screens with every hole 4 μ l, the cell protein obtained after adding 100 μ g different disposal again, mend to cumulative volume 160 μ l/ hole with PBS, be placed in 37 DEG C of lucifuges and cultivate 1h, its fluorescence intensity (excitation wavelength is 380nm, and emission wavelength is 460nm) is detected, each sample parallel three times with fluorescence microplate reader.
(3) results and analysis
As shown in Figure 5, NB group or Cur group reduce the activity of Caspase9 and slightly improve Caspase3 and Caspase8 activity, and compare with NB or Cur group, NB+Cur combination significantly improves Caspase3,8 active and slightly improve the activity of Caspase9, show NB can significantly improve Cur activate Caspase3,8, the activity of 9.
4, ROS Activity determination
(1) experiment material
DHE fluorescent probe
(2) experimental technique
Collect the good logarithmic (log) phase cell of growth conditions, wash 2 times with PBS, centrifugally remove supernatant, add serum-free medium resuspended, counting, adjustment cell density is 1 × 10
6cells/ml, then adding a certain amount of DHE, to make it final concentration be 10 μMs, 30min is hatched in 37 DEG C of incubators, rock once every 5min, after the time of advent, take out cell centrifugation, outwell supernatant, the PBS adding equivalent is resuspended, the NB of variable concentrations is prepared with PBS, Cur and NB+Cur, add in 96 orifice plates with every hole 100 μ l, add again and loaded cell suspension 100 μ l, vibration mixing, the fluorescence intensity in 2h is surveyed with fluorescence microplate reader, excitation wavelength and emission wavelength are respectively 300 and 610nm, using the cell of not agent-feeding treatment as blank, experimental result calculates by 100% of blank group.
(3) results and analysis
As shown in Figure 6, after independent NB process, in its cell, ROS is on a declining curve, and in independent its cell of Cur group, ROS raises slightly, and its ROS of NB and Cur processed group significantly improves, and in time dependence.Experimental result shows, NB can significantly improve the generation of ROS in Cur inducing cell.
Embodiment 440 μ g/mlNB+80 μM of Cur effect A375 cell
Operation is with embodiment 3, and difference is only the difference of Cur concentration, and testing result is as follows:
(1) content detection of curcumin in A375 cell: experimental result shows, contrast blank group, after the process of NB group in its cell curcumin content without significant change, but the content that NB+Cur group but significantly improves Cur in cell is also time dependence.Experimental result (table 3) shows, dextro Borneolum Syntheticum improves curcumin significantly in intracellular absorption.
(2) flow cytometry: experimental result (table 4) shows, compare matched group, obvious Apoptotic cell peak is there is not in NB group, after the process of Cur group, raising appears in Apoptotic cell peak, and compares Cur group, and the apoptosis peak in NB+Cur group improves further, fluidic cell result shows, after adding NB, the subG-1 peak that can significantly improve in Cur inducing cell raises, and shows NB and Cur combined induction apoptosis.
(3) Caspase Activity determination: experimental result (table 5) shows, NB group or Cur group reduce the activity of Caspase9 and slightly improve Caspase3,8 activity, and compare with NB or Cur group, NB+Cur combination significantly improves Caspase3,8 active and slightly improve the activity of Caspase9, show NB can significantly improve Cur activate Caspase3,8, the activity of 9.
(4) ROS Activity determination: experimental result (table 6) shows, after independent NB process, in its cell, ROS is on a declining curve, and in independent its cell of Cur group, ROS raises, and its ROS of NB and Cur processed group significantly improves further, and in time dependence.Experimental result shows, NB can significantly improve the generation of ROS in Cur inducing cell.
The content of curcumin in cell after table 3A375 cell processes different time in different disposal group
Table 4 different disposal group is on the impact of A375 cell cycle
Table 5 different disposal group is on the impact of Caspase activity
Table 6 different disposal group is on the impact of ROS content
Embodiment 540 μ g/mlNB+40 μM of Cur effect A375 cell
Embodiment 5 operates substantially identical with the detection of embodiment 3, and difference is only the difference of Cur concentration, and testing result is as follows:
(1) content detection of curcumin in A375 cell: experimental result (table 7) shows, contrast blank group, in NB group process its cell rear, curcumin content is without significant change, but NB+Cur group but significantly improves the content of Cur in cell and is time dependence.Experimental result shows, dextro Borneolum Syntheticum improves curcumin significantly in intracellular absorption.
(2) flow cytometry: experimental result (table 8) shows, compare matched group, obvious Apoptotic cell peak is there is not in NB group, its SubG-1 is only 2.8%, after the process of Cur group, Apoptotic cell peak brings up to 40.2% from 1.5% of matched group, and compare Cur group, apoptosis peak in NB+Cur group brings up to 86.5% from 40.2%, fluidic cell result shows, after adding NB, the subG-1 peak that can significantly improve in Cur inducing cell raises, and shows NB and Cur combined induction apoptosis.
(3) Caspase Activity determination: shown in experimental result (table 9), NB group or Cur group reduce the activity of Caspase9 and slightly improve Caspase3,8 activity, and compare with NB or Cur group, NB+Cur combination significantly improves Caspase3,8 active and slightly improve the activity of Caspase9, show NB can significantly improve Cur activate Caspase3,8, the activity of 9.
(4) ROS Activity determination: shown in experimental result (table 10), after independent NB process, in its cell, ROS is on a declining curve, in independent its cell of Cur group, ROS raises, and its ROS of NB and Cur processed group significantly improves further, and in time dependence.Experimental result shows, NB can significantly improve the generation of ROS in Cur inducing cell.
The content of curcumin in cell after table 7A375 cell processes different time in different disposal group
Table 8 different disposal group is on the impact of A375 cell cycle
Table 9 different disposal group is on the impact of Caspase activity
Table 10 different disposal group is on the impact of ROS content
Embodiment 6 dextro Borneolum Syntheticum associating curcumin is on the impact of HepG2 cell
1, vitro cell viability detects
(1) experiment material
Hepatocellular carcinoma H22 used in this experiment is purchased from American Type Culture collection warehousing (ATCC, Manassas, VA); Curcumin (Cur), is purchased from Sigma company; Dextro Borneolum Syntheticum (NB), is purchased from Chinese medicine and biological products assay institute; Methyl thiazoly tetrazolium assay (MTT) powder and dimethyl sulfoxide (DMSO), be purchased from Sigma Co., USA; DMEM culture fluid and pancreatin, be purchased from American I nvitrogen (Carlsbad, CA) company.
(2) experimental technique
The HepG2 cell of trophophase of taking the logarithm carries out passage, and adjustment cell density, with 2 × 10
4cells/ml is inoculated in 96 well culture plates, every hole 100 μ l; After 24h, different modes process is carried out to cell, matched group is not for add curcumin and/or dextro Borneolum Syntheticum process, experimental group is as follows: one group adds the curcumin process 24 hours that 100 μ l concentration are respectively 20,40,80 μMs, one group adds the NB process 24 hours that 100 μ l concentration are respectively 20,40,80 μ g/ml, and the 3rd group first adds with the NB process that 50 μ l concentration are respectively 20,40,80 μ g/ml the curcumin that 50 μ l concentration are respectively 20,40,80 μMs again and continue process 24 hours after 12 hours; After 24h, every hole adds MTT (5mg/ml) 20 μ l, puts into incubator and continues to cultivate 4h.The supernatant in hole is carefully sucked after 4h.Every hole adds 150 μ lDMSO dissolving slightly solubility first a ceremonial jade-ladle, used in libations afterwards, and concussion 10min, 570nm survey light absorption value.Calculate cell survival rate, cell survival rate=(OD
experiment-OD
blank)/(OD
contrast-OD
blank) × 100%.Often test in triplicate.
(3) experimental result
As Fig. 7, (concentration in Fig. 7 represents the final concentration of NB and/or Cur in cell in different disposal group; A, b, c, d in bar diagram represent respectively contrast statistically significance analysis between two groups) shown in, add after 20 μ g/mlNB process 12h add 20 μMs of Cur process 24h more maximum to hepatoma carcinoma cell toxicity in the ban.When curcumin (Cur) concentration is 20 μMs, HepG2 cell survival rate is 91.87%, but adding after 20 μ g/mlNB process 12h add 20 μMs of Cur process 24h more in the ban, its cell survival rate drops to 54.25% from 91.87%, have dropped 37.62%.Show, after adding NB, the cytotoxicity of curcumin to HepG2 cell can be significantly improved.
2, flow cytometry
(1) experiment material
PI dyestuff
(2) experimental technique
To take the logarithm the HepG2 cell of trophophase, through pancreatin (routine operation, working solution concentration is 0.25%, lower with) digestion, counting, with 2 × 10
4individual cells/well is inoculated in 6 orifice plates, be placed in incubator (37 DEG C, 5%CO
2) cultivate 24h, then 20 μ g/mlNB100 μ l are added respectively, (NB first processes 12h to 20 μMs of Cur100 μ l and 50 μ l20 μ g/mlNB+50 μ l20 μM of Cur, add Cur process 24h again) process 24h, pump culture medium again, add 1mLPBS (0.01M, pH7.2, down together) cleaning cell removes extracellular dextro Borneolum Syntheticum and curcumin for 3 times, then trypsinization is added, the centrifugal 5min of 1500rpm, collecting cell, add 70% ethanol 1mL of-20 DEG C of pre-coolings, mixing, 4 DEG C are spent the night fixing, next day, the centrifugal 5min of 1500rpm, remove supernatant, 2 times are washed with PBS, add 500 μ l50 μ g/mlPI dye liquors, after after mixing, lucifuge leaves standstill 20min, cross 300 mesh sieves, upper machine testing.10000 cells at least analyzed by each sample.Each experiment in triplicate.
(3) results and analysis
As shown in Figure 8, compare matched group (without NB and/or Cur process), do not occur obvious Apoptotic cell peak in NB group, its SubG-1 is only 0.6%, and G2/M phase cell number is 11.7%; After the process of Cur group, Apoptotic cell peak is 1.8%, and G2/M phase cell number is 31.3%, compare Cur group, apoptosis peak in NB+Cur group does not significantly improve, and be only 4.1%, and G2/M phase cell brings up to 44.6% from 31.3%, after showing to add NB, can significantly improve the G2/M phase cell block in Cur inducing cell, and the number at SubG1 peak does not significantly increase, and shows that NB and Cur carrys out antiproliferative effect by cell death inducing.
3, ROS Activity determination
(1) experiment material
DHE fluorescent probe
(2) experimental technique
Collect the good logarithmic (log) phase HepG2 cell of growth conditions, wash 2 times with PBS, centrifugally remove supernatant, add serum-free medium resuspended, counting, adjustment cell density is 10 × 10
4cells/ml, then adding a certain amount of DHE, to make it final concentration be 10 μMs, 30min is hatched in 37 DEG C of incubators, rock once every 5min, after the time of advent, take out cell centrifugation, outwell supernatant, the PBS adding equivalent is resuspended, the NB of variable concentrations is prepared with PBS, (this group is first with 50 μ lNB process for Cur and NB+Cur, again with 50 μ lCur process, cumulative volume is 100 μ l), add in 96 orifice plates with every hole 100 μ l, add again and loaded cell suspension 100 μ l, vibration mixing, the fluorescence intensity in 2h is surveyed with fluorescence microplate reader, excitation wavelength and emission wavelength are respectively 300 and 610nm, using the cell of not agent-feeding treatment as blank, experimental result calculates by 100% of blank group.Each experiment in triplicate.
(3) results and analysis
As shown in Figure 9, compare matched group (without NB and/or Cur process), in the cell of NB group, ROS is substantially without visible trend, in the cell of Cur group, ROS raises, and comparing Cur group, its ROS of NB and Cur Combined Treatment group improves further, and in time dependence.Experimental result shows, NB can significantly improve the generation of ROS in Cur inducing cell.
Embodiment 7 dextro Borneolum Syntheticum associating demethoxycurcumin is on the impact of HepG2 cell
1, vitro cell viability detects
(1) experiment material
Experiment material used is substantially identical with embodiment 6 step 1, and difference is only: this example uses demethoxycurcumin (DCur is purchased from Sigma company) to replace the curcumin of embodiment 6.
(2) experimental technique
Operation is with embodiment 6 step 1, and difference is, with 1.8 × 10
4cells/ml is inoculated in 96 well culture plates, and uses the corresponding replacement curcumin of demethoxycurcumin.
(3) experimental result
As shown in Figure 10, compare other processed group, add after 20 μ g/mlNB process 12h add 40 μMs of DCur process 24h more maximum to the toxicity of hepatoma carcinoma cell in the ban.When demethoxycurcumin (DCur) concentration is 40 μMs, its cell survival rate is 58.03%, but adding after 20 μ g/mlNB process 12h add 40 μMs of DCur process 24h more in the ban, its cell survival rate drops to 38.69% from 58.03%, have dropped 19.34%.Show, after adding NB, the cytotoxicity of demethoxycurcumin to HepG2 cell can be significantly improved.
2, flow cytometry
(1) experiment material
PI dyestuff
(2) experimental technique
Take the logarithm the HepG2 cell of trophophase, through trypsinization, counting, with 2 × 10
4individual cells/well is inoculated in 6 orifice plates, be placed in incubator (37 DEG C, 5%CO
2) cultivate 24h, then 100 μ l20 μ g/mlNB are added respectively, (NB first processes 12h to 100 μ l40 μM DCur and 50 μ l20 μ g/mlNB+50 μ l40 μM of DCur, add DCur process 24h again) process 24h, pump culture medium again, add 1mLPBS cleaning cell and remove extracellular dextro Borneolum Syntheticum and demethoxycurcumin 3 times, then trypsinization is added, the centrifugal 5min of 1500rpm, collecting cell, add 70% ethanol 1mL of-20 DEG C of pre-coolings, mixing, 4 DEG C are spent the night fixing, next day, the centrifugal 5min of 1500rpm, remove supernatant, 2 times are washed with PBS, add 500 μ lPI dye liquors, after after mixing, lucifuge leaves standstill 20min, cross 300 mesh sieves, upper machine testing.10000 cells at least analyzed by each sample.Each experiment in triplicate.
(3) results and analysis
As shown in figure 11, compare matched group (without NB and/or DCur process), do not occur obvious Apoptotic cell peak in NB group, its SubG-1 is only 0.6%, and G2/M phase cell number is 12.2%; After the process of DCur group, Apoptotic cell peak is 2.2%, and G2/M phase cell number is 28.2%, compare DCur group, apoptosis peak in NB+DCur group does not significantly improve, and be only 2.5%, and G2/M phase cell brings up to 41.3% from 28.2%, after showing to add NB, can significantly improve the G2/M phase cell block in DCur inducing cell, and the number at SubG1 peak does not significantly increase, and shows that NB and DCur carrys out antiproliferative effect by cell death inducing.
3, ROS Activity determination
(1) experiment material
DHE fluorescent probe
(2) experimental technique
Operation is substantially identical with embodiment 1 step 3, and difference is only: this example uses demethoxycurcumin to replace the curcumin of embodiment 1.
(3) results and analysis
As shown in figure 12, compare matched group (without NB and/or DCur process), in the cell of NB group, ROS is substantially without visible trend, in the cell of DCur group, ROS raises, and comparing DCur group, its ROS of NB and DCur Combined Treatment group improves further, and in time dependence.Experimental result shows, NB can significantly improve the generation of ROS in DCur inducing cell.
Embodiment 8 dextro Borneolum Syntheticum associating bisdemethoxycurcumin is on the impact of HepG2 cell
1, vitro cell viability detects
(1) experiment material
Experiment material used is substantially identical with embodiment 6 step 1, and difference is only: this example uses bisdemethoxycurcumin (BDCur is purchased from Sigma company) to replace the curcumin of embodiment 6.
(2) experimental technique
Operation is substantially identical with embodiment 7 step 1, and difference is only: this example uses bisdemethoxycurcumin to replace the demethoxycurcumin of embodiment 7.
(3) experimental result
As shown in figure 13, add after 20 μ g/mlNB process 12h add 40 μMs of BDCur process 24h more maximum to hepatoma carcinoma cell toxicity in the ban.When bisdemethoxycurcumin (BDCur) concentration is 40 μMs, its cell survival rate is 78.98%, but adding after 20 μ g/mlNB process 12h add 40 μMs of BDCur process 24h more in the ban, its cell survival rate drops to 55.41% from 78.98%, have dropped 23.57%.Show, after adding NB, the cytotoxicity of bisdemethoxycurcumin to HepG2 cell can be significantly improved.
2, flow cytometry
(1) experiment material
PI dyestuff
(2) experimental technique
Operation is substantially identical with embodiment 7 step 2, and difference is only: this example uses bisdemethoxycurcumin to replace the demethoxycurcumin of embodiment 7.
(3) results and analysis
As shown in figure 14, compare matched group, do not occur obvious Apoptotic cell peak in NB group, its SubG-1 is only 0.6%, and G2/M phase cell number is 12%; After the process of BDCur group, Apoptotic cell peak is 2.3%, and G2/M phase cell number is 17.5%, compare BDCur group, apoptosis peak in NB+BDCur group does not significantly improve, and be only 1.5%, and G2/M phase cell brings up to 29.5% from 17.5%, after showing to add NB, can significantly improve the G2/M phase cell block in BDCur inducing cell, and the number at SubG1 peak does not significantly increase, and shows that NB and BDCur carrys out antiproliferative effect by cell death inducing.
Three, ROS Activity determination
(1) experiment material
DHE fluorescent probe
(2) experimental technique
Operation is substantially identical with embodiment 6 step 3, and difference is only: this example uses bisdemethoxycurcumin to replace the curcumin of embodiment 6.
(3) results and analysis
As shown in figure 15, compare matched group, in the cell of NB group, ROS is substantially without visible trend, and in the cell of BDCur group, ROS raises, and compares BDCur group, and its ROS of NB and BDCur Combined Treatment group improves further, and in time dependence.Experimental result shows, NB can significantly improve the generation of ROS in BDCur inducing cell.
Above-described embodiment is the present invention's preferably embodiment; but embodiments of the present invention are not restricted to the described embodiments; change, the modification done under other any does not deviate from spirit of the present invention and principle, substitute, combine, simplify; all should be the substitute mode of equivalence, be included within protection scope of the present invention.
Claims (10)
1. dextro Borneolum Syntheticum is as the application of antitumor drug sensitizer.
2. dextro Borneolum Syntheticum according to claim 1 is as the application of antitumor drug sensitizer, it is characterized in that: described antitumor drug is anti-tumor medicine thing.
3. dextro Borneolum Syntheticum according to claim 2 is as the application of antitumor drug sensitizer, it is characterized in that: described anti-tumor medicine thing is at least one in daunorubicin, amycin, darubicin, epirubicin, paclitaxel, lentinan, vincaleucoblastine, vincristine, tamoxifen, Formestane, Anastrozole, flutamide, 5-fluorouracil, methotrexate, cisplatin, carboplatin, oxaliplatin, carmustine, toremifene, ftorafur, curcumin, demethoxycurcumin, bisdemethoxycurcumin and phosphinothioylidynetrisaziridine.
4. dextro Borneolum Syntheticum according to claim 1 is as the application of antitumor drug sensitizer, it is characterized in that: described tumor is hepatocarcinoma, pulmonary carcinoma, malignant melanoma, breast carcinoma, colon cancer, rhinocarcinoma, bladder cancer, cervical cancer, gastric cancer, the esophageal carcinoma or carcinoma of prostate.
5. an antitumor drug sensitizer, is characterized in that: the excipient allowed by dextro Borneolum Syntheticum and medicine or carrier form.
6. an antitumor drug, is characterized in that: be made up of tumour medicine sensitizer according to claim 5 and antitumor drug, composition consists of dextro Borneolum Syntheticum, excipient or carrier and antitumor drug.
7. antitumor drug according to claim 6, is characterized in that: described antitumor drug is anti-tumor medicine thing.
8. antitumor drug according to claim 6, is characterized in that: described anti-tumor medicine thing is at least one in daunorubicin, amycin, darubicin, epirubicin, paclitaxel, lentinan, vincaleucoblastine, vincristine, tamoxifen, Formestane, Anastrozole, flutamide, 5-fluorouracil, methotrexate, cisplatin, carboplatin, oxaliplatin, carmustine, toremifene, ftorafur, curcumin, demethoxycurcumin, bisdemethoxycurcumin and phosphinothioylidynetrisaziridine.
9. antitumor drug according to claim 8, is characterized in that: described antitumor drug is made up of dextro Borneolum Syntheticum, excipient or carrier and curcumin; Described curcumin and described dextro Borneolum Syntheticum 73674 ~ 294696:400000 proportioning in mass ratio.
10. antitumor drug according to claim 8, is characterized in that: described antitumor drug is made up of dextro Borneolum Syntheticum, excipient or carrier and curcumin chemical compounds; Described dextro Borneolum Syntheticum and described curcumin chemical compounds are by quality mol ratio 2 ~ 8 μ g:2 ~ 8nmol proportioning.
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| CN110613703A (en) * | 2018-10-17 | 2019-12-27 | 暨南大学 | Application of d-borneol as adriamycin passivator |
| CN110613704A (en) * | 2018-10-17 | 2019-12-27 | 广东华清园生物科技有限公司 | Application of d-borneol as adriamycin or derivative sensitizer thereof in preparation of anti-lung cancer drugs |
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| WO2017028441A1 (en) * | 2015-08-19 | 2017-02-23 | 华南理工大学 | Application of d-borneol as sensitizer for antitumor drug |
| CN110613703A (en) * | 2018-10-17 | 2019-12-27 | 暨南大学 | Application of d-borneol as adriamycin passivator |
| CN110613704A (en) * | 2018-10-17 | 2019-12-27 | 广东华清园生物科技有限公司 | Application of d-borneol as adriamycin or derivative sensitizer thereof in preparation of anti-lung cancer drugs |
| CN110613704B (en) * | 2018-10-17 | 2023-06-13 | 广东华清园生物科技有限公司 | Application of D-borneol as sensitizer of doxorubicin or derivative thereof in preparation of anti-lung cancer drugs |
| CN112915087A (en) * | 2019-12-05 | 2021-06-08 | 浙江大学 | 5-carboxyl-8-hydroxyquinoline-based antitumor drug sensitizer and application thereof |
| WO2021135550A1 (en) * | 2019-12-31 | 2021-07-08 | 华南理工大学 | Borneol-based polymer, preparation method therefor, and application thereof |
| CN113121810A (en) * | 2019-12-31 | 2021-07-16 | 华南理工大学 | Borneol-based polymer and preparation method and application thereof |
| CN113121810B (en) * | 2019-12-31 | 2022-05-24 | 华南理工大学 | Borneol-based polymer and preparation method and application thereof |
| JP7464949B2 (en) | 2019-12-31 | 2024-04-10 | 華南理工大学 | Borneol-based polymers and their preparation methods and applications |
| CN113362965A (en) * | 2021-06-28 | 2021-09-07 | 中国人民解放军疾病预防控制中心 | System and method for monitoring drug resistance of pathogenic bacteria in hospital |
| CN113362965B (en) * | 2021-06-28 | 2023-09-15 | 中国人民解放军疾病预防控制中心 | System and method for monitoring drug resistance of pathogenic bacteria in hospital |
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