WO2017028441A1 - 右旋龙脑作为抗肿瘤药物增敏剂的应用 - Google Patents

右旋龙脑作为抗肿瘤药物增敏剂的应用 Download PDF

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WO2017028441A1
WO2017028441A1 PCT/CN2015/098535 CN2015098535W WO2017028441A1 WO 2017028441 A1 WO2017028441 A1 WO 2017028441A1 CN 2015098535 W CN2015098535 W CN 2015098535W WO 2017028441 A1 WO2017028441 A1 WO 2017028441A1
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cur
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curcumin
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苏健裕
陈建平
李琳
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South China University of Technology SCUT
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  • the invention relates to the use of right-handed borneol, in particular to the application of right-handed borneol as an anti-tumor drug sensitizer.
  • cancer is mainly based on surgical resection. Because cancer cells are prone to metastasis, surgery cannot be cured, often supplemented by radiotherapy and chemotherapy. Although radiotherapy is more effective than chemotherapy, it also brings a lot of side effects to patients. Therefore, chemical therapy (chemotherapy) is the main means to alleviate the suffering of patients.
  • anti-tumor drugs such as doxorubicin, daunorubicin, cisplatin, 5-fluorouracil, and paclitaxel have been used in clinical practice, bringing good news to patients.
  • chemotherapy also inevitably produces many toxic side effects.
  • Anti-tumor drugs such as doxorubicin and daunorubicin can cause certain toxic side effects to the heart, which can cause sudden tachycardia and acute. Heart failure, difficulty breathing, etc.
  • Paclitaxel can cause allergic reactions after application to the human body, and symptoms such as hypotension, urticaria, and respiratory distress appear. Therefore, how to maximize the anti-tumor effect of drugs and reduce their side effects has always been a hot issue. To this end, it is particularly important to seek a class of chemical sensitizers that not only improve the sensitivity of tumor cells against anti-tumor drugs, but also significantly improve the anti-tumor effect of anti-tumor drugs in therapy.
  • Right-handed borneol is a natural compound extracted from the branches and leaves of the chemical-type plum-blossom of the genus Fructus sinensis. Its chemical structure is as follows:
  • the primary object of the present invention is to overcome the shortcomings and deficiencies of the prior art and to provide the use of dextro-dragon as an anti-tumor drug sensitizer.
  • Another object of the present invention is to provide an antitumor drug sensitizer.
  • the application of the right-handed borneol as an anti-tumor drug sensitizer is to apply the right-handed borneol as an anti-tumor drug sensitizer auxiliary anti-tumor drug.
  • the right-handed dinosaur is the natural right-handed borneol.
  • the antitumor drug is preferably an antitumor chemical.
  • the anti-tumor chemical drugs include: daunorubicin, doxorubicin, demethoxydaunorubicin, epirubicin, paclitaxel, lentinan, vinblastine, vincristine, tamoxifen, formestane, Anastrozole, flutamide, 5-fluorouracil, methotrexate, cisplatin, carboplatin, oxaliplatin, carmustine, toremifene, tegafur, curcumin, demethoxycurcum Ordinary, bis-demethoxycurcumin, thiotepa, etc.; preferably one or at least two of curcumin, demethoxycurcumin and bisdemethoxycurcumin.
  • the tumor mainly refers to non-resistant tumors such as liver cancer, lung cancer, malignant melanoma, breast cancer, colon cancer, nasal cancer, bladder cancer, cervical cancer, gastric cancer, esophageal cancer and prostate cancer.
  • An anti-tumor drug sensitizer is prepared based on the application of D-Dragon brain as an anti-tumor drug sensitizer, which consists of a dextro-borne brain and a drug-allowed excipient or carrier.
  • An anti-tumor drug consisting of a tumor drug sensitizer and an anti-tumor drug, namely, a right-handed borneol, an excipient or a carrier, and an anti-tumor drug.
  • the antitumor drug is preferably an antitumor chemical.
  • the anti-tumor chemical drugs include: daunorubicin, doxorubicin, demethoxydaunorubicin, epirubicin, paclitaxel, lentinan, vinblastine, vincristine, tamoxifen, formestane, Anastrozole, flutamide, 5-fluorouracil, methotrexate, cisplatin, carboplatin, oxaliplatin, carmustine, toremifene, tegafur, curcumin, demethoxycurcum Ordinary, bis-demethoxycurcumin, thiotepa, etc.; preferably one or at least two of paclitaxel, curcumin, demethoxycurcumin and bisdemethoxycurcumin.
  • the tumor mainly refers to a non-resistant tumor, that is, a cancer such as liver cancer, lung cancer, malignant melanoma, breast cancer, colon cancer, nasal cancer, bladder cancer, cervical cancer, gastric cancer, esophageal cancer, and prostate cancer.
  • a cancer such as liver cancer, lung cancer, malignant melanoma, breast cancer, colon cancer, nasal cancer, bladder cancer, cervical cancer, gastric cancer, esophageal cancer, and prostate cancer.
  • the anti-tumor drug preferably consists of dextro-borne brain, an excipient or carrier, and curcumin; it can be used to treat melanoma.
  • the curcumin and the right-handed borneol are matched by a mass ratio of 73674 to 294696:400,000; preferably, the ratio is 73,740:400,000 by mass ratio.
  • the antitumor drug preferably consists of a dextro-borne brain, an excipient or carrier, and a curcuminoid compound; it is useful for treating liver cancer.
  • the curcumin compound is at least one of curcumin, demethoxycurcumin, and bisdemethoxycurcumin.
  • the dextro-borne brain and the curcumin compound are in a molar ratio of 2 to 8 ⁇ g: 2 to 8 nmol.
  • the right-handed borneol and the curcumin are in a molar ratio of 1 ⁇ g: 1 nmol; the right-handed borneol and the demethoxycurcumin are in a molar ratio of 1 ⁇ g: 2 nmol.
  • the present invention has found a new application of dextro-dragon as an anti-tumor chemical sensitizer.
  • Right-handed borneol has low toxicity to normal human cells and has no obvious influence. It is a potential high-efficiency and low-toxic chemical sensitizer.
  • Figure 1 is a graph showing the results of detection of malignant melanoma A375 cells treated with curcumin and curcumin + dextro-dragon.
  • Figure 2 is a graph showing the results of breast cancer cell MCF-7 cells treated with curcumin and curcumin + dextrorone.
  • Figure 3 is a graph showing the effect of different treatment groups on intracellular curcumin content
  • control group indicated that no NB or Cur treatment was added
  • the NB group indicated that 40 ⁇ g/ml NB was added
  • the Cur group indicated that 20 ⁇ M Cur was added
  • the NB+Cur group indicated that 40 ⁇ g/ml NB was added for 12 hours and then 20 ⁇ M of MCur was added.
  • Figure 4 is a graph showing the results of changes in SubG-1 content in A375 cells induced by different treatment groups
  • Figure 5 is a graph showing the results of detection of Caspase 3/8/9 in A375 cells by different treatment groups
  • control group indicated that no NB or Cur treatment was added
  • NB group indicated that 40 ⁇ g/ml NB was added.
  • the Cur group indicates that 20 ⁇ M Cur treatment was added
  • the NB+Cur group indicated that 40 ⁇ g/ml NB was added first for 12 hours and then 20 ⁇ M Cur treatment was added.
  • Figure 6 is a graph showing the results of detection of ROS content in A375 cells induced by different treatment groups
  • the control group indicated that no NB or Cur treatment was added, the NB group indicated that 40 ⁇ g/ml NB was added, the Cur group indicated that 20 ⁇ M Cur was added, and the NB+Cur group indicated that 40 ⁇ g/ml NB was added for 12 hours and then 20 ⁇ M Cur was added.
  • Figure 7 is a graph showing the effect of different treatments of dextro-dragon and curcumin on the survival rate of HepG2 cells; different letters indicate statistically significant differences (P ⁇ 0.05), and the same letters indicate statistically no significant differences. .
  • Figure 8 is a graph showing the effect of different treatments of dextro-dragon and curcumin on the cell cycle of HepG2.
  • Fig. 9 is a graph showing the effect of different treatments of dextro-dragon and curcumin on ROS changes in HepG2 cells.
  • Figure 10 is a graph showing the effect of different treatments of dextrorone and demethoxycurcumin on the survival rate of HepG2 cells; different letters indicate statistically significant differences (P ⁇ 0.05), and the same letters indicate statistically significant There are no significant differences.
  • Figure 11 is a graph showing the effect of different treatments of dextrorone and demethoxycurcumin on the cell cycle of HepG2.
  • Figure 12 is a graph showing the effect of different treatments of dextrorone and demethoxycurcumin on ROS changes in HepG2 cells.
  • Figure 13 is a graph showing the effect of different treatments of dextrorone and bis-demethoxycurcumin on the survival rate of HepG2 cells; different letters indicate statistically significant differences (P ⁇ 0.05), and the same letters indicate statistics. There are no significant differences.
  • Figure 14 is a graph showing the effect of different treatments of dextro-dragon and bis-demethoxycurcumin on the cell cycle of HepG2.
  • Figure 15 is a graph showing the effect of different treatments of dextrorone and bis-demethoxycurcumin on ROS changes in HepG2 cells.
  • Example 1 dextro-dragon brain increases the sensitivity of non-resistant tumor cells to chemical drugs
  • the malignant melanoma cell A375 used in this experiment was purchased from the American model culture collection bank (ATCC, Manassas, VA); the right-handed borneol, China National Institute for Pharmaceutical and Biological Products; curcumin, purchased from Sigma; Methyl methazole blue (MTT) powder and dimethyl sulfoxide (DMSO), Sigma, USA; DMEM medium and trypsin, Invitrogen (Carlsbad, CA), USA; 96-well plate, Corning, USA.
  • ATCC American model culture collection bank
  • VA Manassas, VA
  • curcumin purchased from Sigma
  • DMEM medium and trypsin, Invitrogen Carlsbad, CA
  • 96-well plate Corning, USA.
  • the A375 cells were cultured in DMEM medium containing 10% fetal bovine serum. After 3 days of cell culture, the growth state and cell density of the cells were observed. When the cells were grown to 80%, cell passage was performed. The cells digested with trypsin (conventional operation, working solution concentration: 0.25%, the same below) were transferred to a centrifuge tube, centrifuged at 1000 rpm for 3 min, and the supernatant was discarded, and 10% (v/v) fetal bovine serum was added. 3 ml of DMEM medium, and the gun is sucked more than 10 times. Make sure to resuspend the cells evenly.
  • trypsin conventional operation, working solution concentration: 0.25%, the same below
  • the concentration of D-Dragon brain used in this experiment had no obvious growth inhibition on A375.
  • the concentration of D-Dragon brain was determined to be 40 ⁇ g/ml.
  • the chemical drug Curcumin (Cur) greatly improved the sensitivity of A375 malignant melanoma cells in this experiment, and its sensitivity change index -
  • the dose reduction index (DRI) was 1.72 (see Table 1), indicating that the sensitization effect was significant.
  • Example 2 dextro-dragon brain increases the sensitivity of non-resistant tumor cells to chemical drugs
  • the breast cancer cells MCF-7 used in this experiment were purchased from the American model culture collection library (ATCC, Manassas, VA); the right-handed borneol, China National Institute of Drugs and Biological Products; curcumin, purchased from Sigma; MTT powder and dimethyl sulfoxide (DMSO), Sigma, USA; DMEM medium and trypsin, Invitrogen (Carlsbad, CA), USA; 96-well plate, Corning, USA.
  • ATCC American model culture collection library
  • VA Manassas, VA
  • curcumin purchased from Sigma
  • DMEM medium and trypsin, Invitrogen Carlsbad, CA
  • 96-well plate Corning, USA.
  • the concentration of D-Dragon brain used in this experiment had no obvious growth inhibition on MCF-7.
  • the concentration of D-Dragon brain was determined to be 40 ⁇ g/ml.
  • the chemical drug curcumin (Curcumin) greatly improved the sensitivity of MCF-7 breast cancer cells in this experiment, and its sensitivity change index -
  • the dose reduction index (DRI) was 3.86 (see Table 1), indicating that the sensitization effect was significant.
  • the A375 cells in the logarithmic growth phase were digested with trypsin, counted, and inoculated into a 96-well culture plate at 8 ⁇ 10 4 cells/well, and cultured in an incubator (37 ° C, 5% CO 2 ) for 24 hours.
  • Add 40 ⁇ g/ml NB, 20 ⁇ M Cur and 40 ⁇ g/ml NB+20 ⁇ M Cur (this group was treated with NB for 12h and then add Cur) for 0.5h, 1h, 2h and 4h, then remove the medium and add 1mL PBS.
  • the content of curcumin in the NB group did not change significantly, but the NB+Cur group significantly increased the intracellular Cur content in a time-dependent manner.
  • the Cur content in the NB+Cur group increased from 2.57 in the control group to 3.23, 3.47, 3.99, and 4.36 (10 -9 ⁇ g), respectively, compared with 3.21, 3.40, and 3.55 in the Cur group.
  • 3.86 (10 -9 ⁇ g) increased by 1.01, 1.02, 1.12 and 1.13 times, respectively.
  • the experimental results show that D-Dragon has significantly enhanced the absorption of curcumin in cells.
  • A375 cells in logarithmic growth phase were digested with trypsin, counted, seeded in 6-well plates at 2 ⁇ 10 4 cells/well, and cultured in an incubator (37 ° C, 5% CO 2 ) for 24 h. After adding 40 ⁇ g/ml NB, 20 ⁇ M Cur, and 40 ⁇ g/ml NB+20 ⁇ M Cur (this group was treated with NB for 12 h and then adding Cur), the medium was removed, and the medium was removed by adding 1 mL of PBS to remove the cells 3 times.
  • the cells in the logarithmic growth phase were digested with trypsin, counted, and inoculated into a 10 cm dish at 10 ⁇ 10 4 cells/well, and cultured in an incubator (37 ° C, 5% CO 2 ) for 24 hours.
  • the cells were treated with 40 ⁇ g/ml NB, 20 ⁇ M Cur and 40 ⁇ g/ml NB+20 ⁇ M Cur for 72 h, then the cells were collected and a certain amount of RIPA cell lysate was added (RIPA lysate from 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 %Nonidet P-40 and 0.1% SDS were prepared, depending on the number of cells.
  • the Caspase substrate (Caspase 3, 8, 9) was separately removed, and 4 ⁇ l per well was added to a 96-well fluorescent plate, and then 100 ⁇ g of the cell protein obtained after different treatment was added, and the total volume was adjusted to 160 ⁇ l/well with PBS, and placed at 37.
  • the cells were cultured in the dark for 1 h, and the fluorescence intensity (excitation wavelength was 380 nm, emission wavelength was 460 nm) was measured by a fluorescent microplate reader, and each sample was run in parallel three times.
  • the NB group or the Cur group reduced the activity of Caspase9 and slightly increased the activity of Caspase3 and Caspase8, while the NB+Cur combination significantly increased the activity of Caspase 3 and 8 and slightly increased the Caspase9 compared with the NB or Cur group.
  • Activity indicating that NB can significantly increase the activity of Cur-activated Caspase 3, 8, and 9.
  • Cur and NB+Cur were added to a 96-well plate at 100 ⁇ l per well, and then 100 ⁇ l of the loaded cell suspension was added, shaken and mixed, and the fluorescence intensity within 2 h was measured by a fluorescence microplate reader.
  • the excitation wavelength and emission wavelength were 300 and respectively.
  • cells treated with no drug were used as blank controls, and the results were calculated as 100% of the blank group.
  • Example 5 The detection operation of Example 5 is basically the same as that of Embodiment 3, except that the Cur concentration is different, and the detection results are as follows:
  • the hepatoma cell HepG2 used in this experiment was purchased from the American model culture collection library (ATCC, Manassas, VA); curcumin (Cur), purchased from Sigma; dextro-borne brain (NB), purchased from Chinese medicine and Bioproducts Laboratory; MTT powder and dimethyl sulfoxide (DMSO) were purchased from Sigma, USA; DMEM medium and trypsin were purchased from Invitrogen (Carlsbad, CA).
  • ATCC American model culture collection library
  • Curcumin Curcumin
  • NB dextro-borne brain
  • MTT powder and dimethyl sulfoxide (DMSO) were purchased from Sigma, USA
  • DMEM medium and trypsin were purchased from Invitrogen (Carlsbad, CA).
  • HepG2 cells in logarithmic growth phase were passaged, cell density was adjusted, and seeded in 96-well culture plates at 2 ⁇ 10 4 cells/ml, 100 ⁇ l per well; after 24 h, the cells were treated differently, and the control group was not.
  • the curcumin and/or the right-handed borneol were added.
  • the experimental group was as follows: a group of 100 ⁇ l of curcumin at a concentration of 20, 40, and 80 ⁇ M was treated for 24 hours, and a group of 100 ⁇ l was added at a concentration of 20, 40, and 80 ⁇ g/ml, respectively.
  • FIG. 7 (concentration in Figure 7 indicates the final concentration of NB and/or Cur in the cells in different treatment groups; a, b, c, and d on the bar graph indicate statistically significant differences between the two groups, respectively
  • the liver cancer cells were most toxic after being treated with 20 ⁇ g/ml NB for 12 h and 20 ⁇ M Cur for 24 h.
  • concentration of curcumin (Cur) was 20 ⁇ M
  • the survival rate of HepG2 cells was 91.87%.
  • the cells were treated with 20 ⁇ g/ml NB for 12h and then treated with 20 ⁇ M Cur for 24h, the cell viability was 91.87%. It dropped to 54.25%, a drop of 37.62%. It was shown that the addition of NB significantly increased the cytotoxicity of curcumin on HepG2 cells.
  • HepG2 cells in logarithmic growth phase were digested by trypsin (conventional operation, working solution concentration: 0.25%, the same below), counted, inoculated into 6-well plates at 2 ⁇ 10 4 cells/well, and placed in an incubator.
  • trypsin conventional operation, working solution concentration: 0.25%, the same below
  • the apoptosis peak in the NB+Cur group was not significantly increased, only 4.1%, while the G2/M phase cells increased from 31.3% to 44.6%, indicating that the addition of NB significantly increased the cell cycle of G2/M phase induced by Cur, but the number of SubG1 peaks did not increase significantly, indicating that NB And Cur does not inhibit cell proliferation by inducing apoptosis.
  • the log phase HepG2 cells with good growth status were collected, washed twice with PBS, centrifuged to remove supernatant, resuspended in serum-free medium, counted, adjusted to a cell density of 10 ⁇ 10 4 cells/ml, and then added with a certain amount of DHE.
  • the final concentration was 10 ⁇ M, incubated in a 37 ° C incubator for 30 min, shaking once every 5 min. After the arrival time, the cells were removed for centrifugation, the supernatant was decanted, and the same amount of PBS was resuspended, and PBS was prepared in different concentrations.
  • Cur and NB+Cur (the group was treated with 50 ⁇ l NB and then treated with 50 ⁇ l of Cur, the total volume was 100 ⁇ l), added to a 96-well plate at 100 ⁇ l per well, and then 100 ⁇ l of the loaded cell suspension was added, and the mixture was shaken and mixed. Fluorescence intensity was measured by fluorescence microplate reader for 2 hours, and the excitation wavelength and emission wavelength were 300 and 610 nm, respectively. The cells without drug treatment were used as blank control, and the experimental results were calculated according to 100% of the blank group. Repeat three times for each experiment.
  • Step 1 of Example 6 The procedure was the same as in Step 1 of Example 6, except that it was inoculated into a 96-well culture plate at 1.8 ⁇ 10 4 cells/ml, and the curcumin was replaced with demethoxycurcumin.
  • the toxicity of liver cancer cells was the highest after treatment with 20 ⁇ g/ml NB for 12 h and 40 ⁇ M DCur for 24 h.
  • concentration of demethoxycurcumin (DCur) was 40 ⁇ M
  • the cell viability was 58.03%.
  • the cell viability decreased from 58.03% to 38.69. %, down 19.34%. It was shown that the addition of NB significantly increased the cytotoxicity of demethoxycurcumin on HepG2 cells.
  • HepG2 cells in logarithmic growth phase were digested with trypsin, counted, seeded in 6-well plates at 2 ⁇ 10 4 cells/well, and placed in an incubator (37 ° C, 5% CO 2 ) for 24 h, respectively.
  • the liver cancer cells were most toxic after being treated with 20 ⁇ g/ml NB for 12 h and 40 ⁇ M BDCur for 24 h.
  • concentration of bis-demethoxycurcumin (BDCur) was 40 ⁇ M
  • the cell viability was 78.98%.
  • the cell viability decreased from 78.98% to 55.41%, down by 23.57%. Indicating that Tim After adding NB, the cytotoxicity of bis-demethoxycurcumin on HepG2 cells was significantly increased.
  • the intracellular ROS of the NB group had no obvious trend, and the intracellular ROS of the BDCur group increased, whereas compared with the BDCur group, the NB and BDCur combined treatment group had further ROS. Increased and time dependent.
  • the experimental results show that NB can significantly increase the production of ROS in BDCur induced cells.

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Abstract

本发明提供了右旋龙脑在制备抗肿瘤药物增敏剂中的应用。当使用对细胞生长没有明显影响的右旋龙脑浓度后，非耐药细胞对抗肿瘤化学药物的敏感性增强。抗肿瘤化学药物浓度降低，毒副作用减少，而疗效没有降低。

Description

右旋龙脑作为抗肿瘤药物增敏剂的应用 技术领域
本发明涉及右旋龙脑的用途，特别涉及右旋龙脑作为抗肿瘤药物增敏剂的应用。
背景技术
目前，癌症的治疗主要是以手术切除为主，由于癌细胞易发生转移，故手术并不能根治，常常辅以放疗和化疗。虽然放疗比化疗作用效果快，但也给病人带来了很多的副作用，因此，化学药物治疗(化疗)被寄以是减轻病人痛苦的主要手段。
半个世纪以来，肿瘤化疗有了迅速的发展。各种抗肿瘤药物如阿霉素、柔红霉素、顺铂、5-氟尿嘧啶、紫杉醇等相继应用于临床，给病人带来了福音。然而，化疗也不可避免地会产生许多的毒副作用，如阿霉素和柔红霉素等抗肿瘤药物应用于人体之后会给心脏带来一定的毒副作用，可以引起突发心动过速、急性心力衰竭、呼吸困难等。紫杉醇应用人体之后会引起过敏反应，出现低血压、荨麻疹、呼吸窘迫等症状。因此，如何最大程度的发挥药物的抗肿瘤效果又能降低其毒副作用一直以来是研究的热点问题。为此，寻求一类不仅能提高肿瘤细胞对抗肿瘤药物的敏感性，而且能显著提高抗肿瘤药物在治疗中的抑瘤效果的化学药物增敏剂就显得尤为的重要。
右旋龙脑是一种从樟科阴香化学型梅片树枝叶中提取得到的天然化合物，其化学结构式如下：
Figure PCTCN2015098535-appb-000001
众所周知，右旋龙脑用于闭证神昏、目赤肿痛、喉痹口疮、疮疡肿痛，溃后不敛和心绞痛等。目前，尚未有关右旋龙脑具有抗肿瘤药物增敏剂功能的报道。
发明内容
本发明的首要目的在于克服现有技术的缺点与不足，提供右旋龙脑作为抗肿瘤药物增敏剂的应用。
本发明的另一目的在于提供一种抗肿瘤药物增敏剂。
本发明的再一目的在于提供一种抗肿瘤药物。
本发明的目的通过下述技术方案实现：右旋龙脑作为抗肿瘤药物增敏剂的应用，是将右旋龙脑作为抗肿瘤药物增敏剂辅助抗肿瘤药物进行应用。
所述的右旋龙脑即为天然右旋龙脑。
所述的抗肿瘤药物优选为抗肿瘤化学药物。
所述的抗肿瘤化学药物包括：柔红霉素、阿霉素、去甲氧柔红霉素、表阿霉素、紫杉醇、香菇多糖、长春花碱、长春新碱、三苯氧胺、福美司坦、阿那曲唑、氟他胺、5-氟尿嘧啶、甲氨蝶呤、顺铂、卡铂、奥沙利铂、卡莫司汀、托瑞米芬、替加氟、姜黄素、去甲氧基姜黄素、双去甲氧基姜黄素和塞替派等；优选为姜黄素、去甲氧基姜黄素和双去甲氧基姜黄素中的一种或至少两种。
所述的肿瘤主要指的是非耐药肿瘤，如肝癌、肺癌、恶性黑色瘤、乳腺癌、结肠癌、鼻癌、膀胱癌、宫颈癌、胃癌、食管癌和前列腺癌。
一种抗肿瘤药物增敏剂，是基于右旋龙脑作为抗肿瘤药物增敏剂的应用制备得到，其由右旋龙脑和药物允许的赋形剂或载体组成。
一种抗肿瘤药物，由肿瘤药物增敏剂和抗肿瘤药物组成，即由右旋龙脑、赋形剂或载体、和抗肿瘤药物组成。
所述的抗肿瘤药物优选为抗肿瘤化学药物。
所述的抗肿瘤化学药物包括：柔红霉素、阿霉素、去甲氧柔红霉素、表阿霉素、紫杉醇、香菇多糖、长春花碱、长春新碱、三苯氧胺、福美司坦、阿那曲唑、氟他胺、5-氟尿嘧啶、甲氨蝶呤、顺铂、卡铂、奥沙利铂、卡莫司汀、托瑞米芬、替加氟、姜黄素、去甲氧基姜黄素、双去甲氧基姜黄素和塞替派等；优选为紫杉醇、姜黄素、去甲氧基姜黄素和双去甲氧基姜黄素中的一种或至少两种。
所述的肿瘤主要指的是非耐药肿瘤，即癌症，如肝癌、肺癌、恶性黑色瘤、乳腺癌、结肠癌、鼻癌、膀胱癌、宫颈癌、胃癌、食管癌和前列腺癌。
所述的抗肿瘤药物优选由右旋龙脑、赋形剂或载体、和姜黄素组成；其可用于治疗黑色素瘤。
所述的姜黄素与所述的右旋龙脑按质量比73674～294696：400000配比；优选按质量比73674：400000配比。
所述的抗肿瘤药物优选由右旋龙脑、赋形剂或载体、和姜黄素类化合物组成；其可用于治疗肝癌。
所述的姜黄素类化合物为姜黄素、去甲氧基姜黄素和双去甲氧基姜黄素中的至少一种。
所述的右旋龙脑和所述的姜黄素类化合物按质量摩尔比2～8μg：2～8nmol配比。
优选地，所述的右旋龙脑和所述的姜黄素按质量摩尔比1μg：1nmol配比；所述的右旋龙脑和所述的去甲氧基姜黄素按质量摩尔比1μg：2nmol配比；所述的右旋龙脑和所述的双去甲氧基姜黄素按质量摩尔比1μg：2nmol配比。
本发明相对于现有技术具有如下的优点及效果：
(1)本发明发现了右旋龙脑作为抗肿瘤化学药物增敏剂的新应用。
(2)在加用了对细胞无明显生长影响的右旋龙脑浓度后，非耐药细胞对抗肿瘤化学药物的敏感性大大增加，即在加用右旋龙脑的基础上，可将用于杀伤肿瘤细胞的抗肿瘤化学药物的浓度降低，但疗效并没有降低。而化学药物的毒副作用随着其浓度的降低而减少，保护人体正常细胞。
(3)右旋龙脑对人体正常细胞毒性低，基本无明显影响，是一种潜在的高效低毒的化学药物增敏剂。
附图说明
图1是恶性黑色素瘤A375细胞分别经过姜黄素和姜黄素+右旋龙脑处理后的检测结果图。
图2是乳腺癌细胞MCF-7细胞分别经过姜黄素和姜黄素+右旋龙脑处理后的检测结果图。
图3是不同处理组对细胞内姜黄素含量的影响结果图；
其中：对照组表示不添加NB或Cur处理，NB组表示添加40μg/ml NB处理，Cur组表示添加20μM Cur处理，NB+Cur组表示先添加40μg/ml NB处理12h后添加20μMCur处理。
图4是不同处理组诱个导A375细胞内SubG-1含量的变化结果图；
其中，“-”表示未添加，“+”表示添加。
图5是不同处理组激活A375细胞内Caspase3/8/9的检测结果图；
其中，对照组表示不添加NB或Cur处理，NB组表示添加40μg/ml NB处理， Cur组表示添加20μM Cur处理，NB+Cur组表示先添加40μg/ml NB处理12h后添加20μM Cur处理。
图6是不同处理组诱导A375细胞内ROS含量的检测结果图；
其中，对照组表示不添加NB或Cur处理，NB组表示添加40μg/ml NB处理，Cur组表示添加20μM Cur处理，NB+Cur组表示先添加40μg/ml NB处理12h后添加20μM Cur处理。
图7是右旋龙脑与姜黄素的不同处理方式对HepG2细胞存活率的影响结果图；不同字母表示统计学上有显著性差异(P<0.05)，相同字母表示统计学上没有显著性差异。
图8是右旋龙脑与姜黄素的不同处理方式对HepG2细胞周期的影响结果图。
图9右旋龙脑与姜黄素的不同处理方式对HepG2细胞内ROS变化的影响结果图。
图10是右旋龙脑与去甲氧基姜黄素的不同处理方式对HepG2细胞存活率的影响结果图；不同字母表示统计学上有显著性差异(P<0.05)，相同字母表示统计学上没有显著性差异。
图11是右旋龙脑与去甲氧基姜黄素的不同处理方式对HepG2细胞周期的影响结果图。
图12是右旋龙脑与去甲氧基姜黄素的不同处理方式对HepG2细胞内ROS变化的影响结果图。
图13是右旋龙脑与双去甲氧基姜黄素的不同处理方式对HepG2细胞存活率的影响结果图；不同字母表示统计学上有显著性差异(P<0.05)，相同字母表示统计学上没有显著性差异。
图14是右旋龙脑与双去甲氧基姜黄素的不同处理方式对HepG2细胞周期的影响结果图。
图15是右旋龙脑与双去甲氧基姜黄素的不同处理方式对HepG2细胞内ROS变化的影响结果图。
具体实施方式
下面结合实施例及附图对本发明作进一步详细的描述，但本发明的实施方式不限于此。
实施例1右旋龙脑增加非耐药肿瘤细胞对化学药物的敏感性
(1)实验材料
本实验中所用的恶性黑色素瘤细胞A375购自于美国模式培养物集存库(ATCC，Manassas,VA)；右旋龙脑，中国药品与生物制品检定所；姜黄素，购于Sigma公司；四甲基偶氮唑蓝(MTT)粉末及二甲基亚砜(DMSO)，美国Sigma公司；DMEM培养液及胰酶，美国Invitrogen(Carlsbad,CA)公司；96孔板，美国Corning公司。
(2)实验方法
将A375细胞培养于含体积百分比10％胎牛血清的DMEM培养液中，细胞培养3天后，观察细胞的生长状态和细胞密度，当细胞长到80％时，进行细胞传代。将胰酶(常规操作，工作液浓度为0.25％，下同)消化下后的细胞转移至离心管中，1000rpm离心3min后弃去上清，加含10％(v/v)胎牛血清的DMEM培养基3ml，枪吸打10次以上，务必使细胞重悬均匀，取10μl细胞混悬液台盼蓝染色计活细胞数后用新鲜培养基调整细胞密度为1.8×104cells/ml接种于96孔培养板中，每孔100μl；24h后，一组加入不同浓度的化学药物100μl，另一组加40μg/mL右旋龙脑50μl和不同浓度化学药物的50μl。72h之后，在显微镜下拍照观察细胞的生长状态。然后每孔加入MTT(5mg/ml)20μl，放入培养箱中继续培养4h。4h后小心吸去孔内的上清液。之后每孔加入150μl的DMSO，震荡10min，490nm测吸光值。计算细胞存活率，细胞存活率＝(OD实验-OD空白)/(OD对照-OD空白)×100％。计算好后，将不同处理组与细胞存活率做柱状图，并做t-检验进行显著性差异分析实验结果。用origin 8.0软件计算半数抑制率(IC50)，并按公式计算剂量下降指数(Dose Reduction Index，DRI)＝单用某化学药物后对肿瘤细胞的IC50/用化学药物加右旋龙脑后对肿瘤细胞的IC50。每实验重复三次。
(3)实验结果
本实验采用的右旋龙脑浓度对A375无明显生长抑制。经预实验，确定了右旋龙脑的浓度为40μg/ml。如图1所示，在加用了浓度为40μg/ml的右旋龙脑之后，化学药物姜黄素(Curcumin，Cur)对本实验的A375恶性黑色素瘤细胞的敏感性大大提高，其敏感性变化指标-剂量下降指数(DRI)为1.72(见表1)，说明增敏效果明显。
表1 加用右旋龙脑后姜黄素对A375细胞的IC50和DRI的变化
Figure PCTCN2015098535-appb-000002
实施例2右旋龙脑增加非耐药肿瘤细胞对化学药物的敏感性
(1)实验材料
本实验中所用的乳腺癌细胞MCF-7购自于美国模式培养物集存库(ATCC，Manassas,VA)；右旋龙脑，中国药品与生物制品检定所；姜黄素，购于Sigma公司；四甲基偶氮唑蓝(MTT)粉末及二甲基亚砜(DMSO)，美国Sigma公司；DMEM培养液及胰酶，美国Invitrogen(Carlsbad,CA)公司；96孔板，美国Corning公司。
(2)实验方法
操作同实施例1。
(3)实验结果
本实验采用的右旋龙脑浓度对MCF-7无明显生长抑制。经预实验，确定了右旋龙脑的浓度为40μg/ml。如图2所示，在加用了浓度为40μg/ml的右旋龙脑之后，化学药物姜黄素(Curcumin)对本实验的MCF-7乳腺癌细胞的敏感性大大提高，其敏感性变化指标-剂量下降指数(DRI)为3.86(见表1)，说明增敏效果明显。
表2 加用右旋龙脑后姜黄素对MCF-7细胞的IC50和DRI的变化
Figure PCTCN2015098535-appb-000003
实施例3 40μg/ml NB+20μM Cur作用A375细胞
1、A375细胞内姜黄素的含量检测
(1)实验材料
含1％(v/v)Trition X-100的NaOH溶液(NaOH的浓度为0.1mol/L)，简称细胞裂解液。
(2)实验方法
取对数生长期的A375细胞，经胰酶消化，计数，以8×104个细胞/孔接种于96孔培养板，置于培养箱中(37℃，5％CO2)培养24h后。分别加入40μg/ml NB、20μM Cur以及40μg/ml NB+20μM Cur(该组为先用NB处理12h后再加入Cur)处理0.5h、1h、2h和4h后，抽去培养基，加入1mL PBS(0.01M、pH7.2，下同)清洗细胞3遍除去细胞外的右旋龙脑和姜黄素，然后加入200μL细胞裂解液，振荡10min后，在荧光酶标仪下进行姜黄素含量的测定。其中，激发波长为425nm，发射波长为545nm。进行三次重复实验。
(3)实验结果与分析
如图3所示，对比空白对照组，NB组处理后其细胞内姜黄素含量无明显变化，可是NB+Cur组却显著提高了细胞内Cur的含量并呈时间依赖性。在0.5、1、2、4h后，NB+Cur组中Cur的含量分别从对照组的2.57提高到3.23、3.47、3.99和4.36(10-9μg)，比Cur组的3.21、3.40、3.55和3.86(10-9μg)分别提高了1.01、1.02、1.12和1.13倍。实验结果表明，右旋龙脑显著的提高了姜黄素在细胞内的吸收。
2、流式细胞分析
(1)实验材料
PI染料(浓度是50μg/ml)
(2)实验方法
取对数生长期的A375细胞，经胰酶消化，计数，以2×104个细胞/孔接种于6孔板，置于培养箱中(37℃，5％CO2)培养24h后。分别加入40μg/ml NB、20μM Cur、以及40μg/ml NB+20μM Cur(该组为先用NB处理12h后再加入Cur)处理72h后，抽去培养基，加入1mL PBS清洗细胞3遍除去细胞外的右旋龙脑和姜黄素，然后加入胰酶消化，1500rpm离心5min，收集细胞，加入-20℃预冷的70％乙醇1mL，混匀，4℃过夜固定，次日，1500rpm离心5min，去上清，用PBS洗2遍，加500μl PI染液，混匀后避光静置20min后，过300目筛，上机检测。每个样品至少分析10000个细胞。
(3)结果与分析
如图4所示，相比对照组而言，NB组内并没有出现明显的凋亡细胞峰，其SubG-1仅为2.5％，Cur组处理后凋亡细胞峰从对照组的1.4％提高到27.5％，而相比Cur组，NB+Cur组中的细胞凋亡峰从27.5％提高到75％，流式细胞结果表明，加入NB后，能显著提高Cur诱导细胞内的subG-1峰升高，表明NB和Cur联合诱导细胞凋亡。
3、Caspase(含半胱氨酸的天冬氨酸蛋白水解酶)活性检测
(1)实验材料
Caspase底物Caspase3、Caspase8、Caspase9
(2)实验方法
取对数生长期的细胞，经胰酶消化，计数，以10×104个细胞/孔接种于10cm皿中，置于培养箱中(37℃，5％CO2)培养24h后。分别加入40μg/ml NB、20μM Cur以及40μg/ml NB+20μM Cur处理72h，然后收集细胞，加入一定量的RIPA细胞裂解液(RIPA裂解液由50mM Tris-HCl(pH 7.4)、150mM NaCl,1％Nonidet P-40和0.1％SDS配制而成，添加量视细胞数目而定)，冰上孵育1h后， 在11000g下离心30min，取上清液转移到新的EP管中。然后采用BCA试剂盒测定蛋白浓度。然后分别移取Caspase底物(Caspase3、8、9)，以每孔4μl加入96孔荧光板中，再加入100μg不同处理后获得的细胞蛋白，用PBS补至总体积160μl/孔，置于37℃避光培养1h，用荧光酶标仪检测其荧光强度(激发波长为380nm，发射波长为460nm)，每个样品平行三次。
(3)结果与分析
如图5所示，NB组或Cur组降低了Caspase9的活性和轻微提高了Caspase3和Caspase8活性，而与NB或Cur组比较，NB+Cur组合显著提高了Caspase3、8活性和轻微提高了Caspase9的活性，表明NB能显著提高Cur激活Caspase3、8、9的活性。
4、ROS活性检测
(1)实验材料
DHE荧光探针
(2)实验方法
收集生长状态良好的对数期细胞，用PBS洗2次，离心去上清，加无血清培养基重悬，计数，调整细胞密度为1×106cells/ml，然后加入一定量的DHE使之终浓度为10μM，于37℃培养箱孵育30min，每隔5min摇晃一次，到达时间后，取出细胞离心，倒掉上清液，加等量的PBS重悬，用PBS配制不同浓度的NB、Cur和NB+Cur，以每孔100μl加入96孔板中，再加入已装载细胞悬液100μl，振荡混匀，用荧光酶标仪测2h内的荧光强度，激发波长和发射波长分别为300和610nm，以不加药处理的细胞作为空白对照，实验结果按空白组的100％计算。
(3)结果与分析
如图6所示，单独NB处理后其细胞内ROS呈下降趋势，单独Cur组其细胞内ROS略微升高，而NB和Cur处理组其ROS显著提高，并呈时间依赖性。实验结果表明，NB能显著提高Cur诱导细胞内ROS的产生。
实施例4 40μg/ml NB+80μM Cur作用A375细胞
操作同实施例3，区别仅在于Cur浓度的不同，检测结果如下：
(1)A375细胞内姜黄素的含量检测：实验结果显示，对比空白对照组，NB组处理后其细胞内姜黄素含量无明显变化，可是NB+Cur组却显著提高了细胞内Cur的含量并呈时间依赖性。实验结果(表3)表明，右旋龙脑显著的提高了姜黄素在细胞内的吸收。
(2)流式细胞分析：实验结果(表4)显示，相比对照组而言，NB组内并没有出现明显的凋亡细胞峰，Cur组处理后凋亡细胞峰出现升高，而相比Cur组， NB+Cur组中的细胞凋亡峰进一步提高，流式细胞结果表明，加入NB后，能显著提高Cur诱导细胞内的subG-1峰升高，表明NB和Cur联合诱导细胞凋亡。
(3)Caspase活性检测：实验结果(表5)显示，NB组或Cur组降低了Caspase9的活性和轻微提高了Caspase3、8活性，而与NB或Cur组比较，NB+Cur组合显著提高了Caspase 3、8活性和轻微提高了Caspase 9的活性，表明NB能显著提高Cur激活Caspase 3、8、9的活性。
(4)ROS活性检测：实验结果(表6)显示，单独NB处理后其细胞内ROS呈下降趋势，单独Cur组其细胞内ROS升高，而NB和Cur处理组其ROS进一步显著提高，并呈时间依赖性。实验结果表明，NB能显著提高Cur诱导细胞内ROS的产生。
表3 A375细胞在不同处理组中处理不同时间后细胞内姜黄素的含量
Figure PCTCN2015098535-appb-000004
表4 不同处理组对A375细胞周期的影响
Figure PCTCN2015098535-appb-000005
表5 不同处理组对Caspase活性的影响
Figure PCTCN2015098535-appb-000006
表6 不同处理组对ROS含量的影响
Figure PCTCN2015098535-appb-000007
实施例5 40μg/ml NB+40μM Cur作用A375细胞
实施例5与实施例3的检测操作基本相同，区别仅在于Cur浓度的不同，检测结果如下：
(1)A375细胞内姜黄素的含量检测：实验结果(表7)显示，对比空白对照组，NB组处理后其细胞内姜黄素含量无明显变化，可是NB+Cur组却显著提高了细胞内Cur的含量并呈时间依赖性。实验结果表明，右旋龙脑显著的提高了姜黄素在细胞内的吸收。
(2)流式细胞分析：实验结果(表8)显示，相比对照组而言，NB组内并没有出现明显的凋亡细胞峰，其SubG-1仅为2.8％，Cur组处理后凋亡细胞峰从对照组的1.5％提高到40.2％，而相比Cur组，NB+Cur组中的细胞凋亡峰从40.2％提高到86.5％，流式细胞结果表明，加入NB后，能显著提高Cur诱导细胞内的subG-1峰升高，表明NB和Cur联合诱导细胞凋亡。
(3)Caspase活性检测：实验结果(表9)所示，NB组或Cur组降低了Caspase 9的活性和轻微提高了Caspase 3、8活性，而与NB或Cur组比较，NB+Cur组合显著提高了Caspase 3、8活性和轻微提高了Caspase 9的活性，表明NB能显著提高Cur激活Caspase 3、8、9的活性。
(4)ROS活性检测：实验结果(表10)所示，单独NB处理后其细胞内ROS呈下降趋势，单独Cur组其细胞内ROS升高，而NB和Cur处理组其ROS进一步显著提高，并呈时间依赖性。实验结果表明，NB能显著提高Cur诱导细胞内ROS的产生。
表7 A375细胞在不同处理组中处理不同时间后细胞内姜黄素的含量
Figure PCTCN2015098535-appb-000008
表8 不同处理组对A375细胞周期的影响
Figure PCTCN2015098535-appb-000009
表9 不同处理组对Caspase活性的影响
Figure PCTCN2015098535-appb-000010
表10 不同处理组对ROS含量的影响
Figure PCTCN2015098535-appb-000011
实施例6右旋龙脑联合姜黄素对HepG2细胞的影响
1、体外细胞存活率检测
(1)实验材料
本实验中所用的肝癌细胞HepG2购自于美国模式培养物集存库(ATCC，Manassas,VA)；姜黄素(Cur)，购于Sigma公司；右旋龙脑(NB)，购于中国药品与生物制品检定所；四甲基偶氮唑蓝(MTT)粉末及二甲基亚砜(DMSO)，购于美国Sigma公司；DMEM培养液及胰酶，购于美国Invitrogen(Carlsbad,CA)公司。
(2)实验方法
取对数生长期的HepG2细胞进行细胞传代，调整细胞密度，以2×104cells/ml接种于96孔培养板中，每孔100μl；24h后，对细胞进行不同方式处理，对照组为不添加姜黄素和/或右旋龙脑处理，实验组如下：一组加入100μl浓度分别为20、40、80μM的姜黄素处理24小时，一组加入100μl浓度分别为20、40、80μg/ml的NB处理24小时，第三组先用50μl浓度分别为20、40、80μg/ml的NB处理12小时后再加入50μl浓度分别为20、40、80μM的姜黄素继续处理24小时；24h后，每孔加入MTT(5mg/ml)20μl，放入培养箱中继续培养4h。4h后小心吸去孔内的上清液。之后每孔加入150μl DMSO溶解难溶性甲瓒，震荡10min，570nm测吸光值。计算细胞存活率，细胞存活率＝(OD实验-OD空白)/(OD对照-OD空白)×100％。每实验重复三次。
(3)实验结果
如图7(图7中的浓度表示不同处理组中NB和/或Cur在细胞中的终浓度；柱形图上的a、b、c、d分别表示两组之间对比统计学上显著性分析)所示，当先加入20μg/ml NB处理12h再加20μM Cur处理24h之后对肝癌细胞毒性最大。当姜黄素(Cur)浓度为20μM时，HepG2细胞存活率为91.87％，然而当先加入20μg/ml NB处理12h再加20μM Cur处理24h之后，其细胞存活率从91.87％下 降到54.25％，下降了37.62％。表明，添加NB之后，能显著提高姜黄素对HepG2细胞的细胞毒性。
2、流式细胞分析
(1)实验材料
PI染料
(2)实验方法
取对数生长期的HepG2细胞，经胰酶(常规操作，工作液浓度为0.25％，下同)消化，计数，以2×104个细胞/孔接种于6孔板，置于培养箱中(37℃，5％CO2)培养24h，接着分别加入20μg/ml NB 100μl、20μM Cur 100μl和50μl 20μg/ml NB+50μl 20μM Cur(NB先处理12h，再加入Cur处理24h)处理24h，再抽去培养基，加入1mL PBS(0.01M、pH7.2，下同)清洗细胞3遍除去细胞外的右旋龙脑和姜黄素，然后加入胰酶消化，1500rpm离心5min，收集细胞，加入-20℃预冷的70％乙醇1mL，混匀，4℃过夜固定，次日，1500rpm离心5min，去上清，用PBS洗2遍，加500μl 50μg/ml PI染液，混匀后避光静置20min后，过300目筛，上机检测。每个样品至少分析10000个细胞。每次实验重复三次。
(3)结果与分析
如图8所示，相比对照组(不用NB和/或Cur处理)而言，NB组内并没有出现明显的凋亡细胞峰，其SubG-1仅为0.6％，且G2/M期细胞数目为11.7％；Cur组处理后凋亡细胞峰为1.8％，且G2/M期细胞数目为31.3％，相比Cur组，NB+Cur组中的细胞凋亡峰并没有显著提高，仅为4.1％，而G2/M期细胞从31.3％提高到44.6％，表明加入NB后，能显著提高Cur诱导细胞内的G2/M期细胞阻滞，而SubG1峰的数目并没有显著增加，表明NB和Cur并不是通过诱导细胞凋亡来抑制细胞增殖。
3、ROS活性检测
(1)实验材料
DHE荧光探针
(2)实验方法
收集生长状态良好的对数期HepG2细胞，用PBS洗2次，离心去上清，加无血清培养基重悬，计数，调整细胞密度为10×104cells/ml，然后加入一定量的DHE使之终浓度为10μM，于37℃培养箱孵育30min，每隔5min摇晃一次，到达时间后，取出细胞离心，倒掉上清液，加等量的PBS重悬，用PBS配制不同浓度的NB、Cur和NB+Cur(该组先用50μl NB处理，再用50μl Cur处理，总体积为100μl)，以每孔100μl加入96孔板中，再加入已装载细胞悬液100μl，振荡混匀，用荧光酶标仪测2h内的荧光强度，激发波长和发射波长分别为300 和610nm，以不加药处理的细胞作为空白对照，实验结果按空白组的100％计算。每次实验重复三次。
(3)结果与分析
如图9所示，相比对照组(不用NB和/或Cur处理)来说，NB组的细胞内ROS基本无明显趋势，Cur组的细胞内ROS升高，而相比Cur组而言，NB和Cur联合处理组其ROS进一步提高，并呈时间依赖性。实验结果表明，NB能显著提高Cur诱导细胞内ROS的产生。
实施例7右旋龙脑联合去甲氧基姜黄素对HepG2细胞的影响
1、体外细胞存活率检测
(1)实验材料
所用的实验材料大体与实施例6步骤1相同，区别仅在于：本实例使用去甲氧基姜黄素(DCur，购于Sigma公司)取代实施例6的姜黄素。
(2)实验方法
操作同实施例6步骤1，区别在于，以1.8×104cells/ml接种于96孔培养板中，以及使用去甲氧基姜黄素相应取代姜黄素。
(3)实验结果
如图10所示，相比其他处理组，当先加入20μg/ml NB处理12h再加40μM DCur处理24h之后对肝癌细胞的毒性最大。当去甲氧基姜黄素(DCur)浓度为40μM时，其细胞存活率为58.03％，然而当先加入20μg/ml NB处理12h再加40μM DCur处理24h之后，其细胞存活率从58.03％下降到38.69％，下降了19.34％。表明，添加NB之后，能显著提高去甲氧基姜黄素对HepG2细胞的细胞毒性。
2、流式细胞分析
(1)实验材料
PI染料
(2)实验方法
取对数生长期的HepG2细胞，经胰酶消化，计数，以2×104个细胞/孔接种于6孔板，置于培养箱中(37℃，5％CO2)培养24h，接着分别加入100μl 20μg/ml NB、100μl 40μM DCur和50μl 20μg/ml NB+50μl 40μM DCur(NB先处理12h，再加入DCur处理24h)处理24h，再抽去培养基，加入1mL PBS清洗细胞3遍除去细胞外的右旋龙脑和去甲氧基姜黄素，然后加入胰酶消化，1500rpm离心5min，收集细胞，加入-20℃预冷的70％乙醇1mL，混匀，4℃过夜固定，次日，1500rpm离心5min，去上清，用PBS洗2遍，加500μl PI染液，混匀后避光静置20min后，过300目筛，上机检测。每个样品至少分析10000 个细胞。每次实验重复三次。
(3)结果与分析
如图11所示，相比对照组(不用NB和/或DCur处理)而言，NB组内并没有出现明显的凋亡细胞峰，其SubG-1仅为0.6％，且G2/M期细胞数目为12.2％；DCur组处理后凋亡细胞峰为2.2％，且G2/M期细胞数目为28.2％，相比DCur组，NB+DCur组中的细胞凋亡峰并没有显著提高，仅为2.5％，而G2/M期细胞从28.2％提高到41.3％，表明加入NB后，能显著提高DCur诱导细胞内的G2/M期细胞阻滞，而SubG1峰的数目并没有显著增加，表明NB和DCur并不是通过诱导细胞凋亡来抑制细胞增殖。
3、ROS活性检测
(1)实验材料
DHE荧光探针
(2)实验方法
操作大体与实施例1步骤三相同，区别仅在于：本实例使用去甲氧基姜黄素取代实施例1的姜黄素。
(3)结果与分析
如图12所示，相比对照组(不用NB和/或DCur处理)来说，NB组的细胞内ROS基本无明显趋势，DCur组的细胞内ROS升高，而相比DCur组而言，NB和DCur联合处理组其ROS进一步提高，并呈时间依赖性。实验结果表明，NB能显著提高DCur诱导细胞内ROS的产生。
实施例8右旋龙脑联合双去甲氧基姜黄素对HepG2细胞的影响
1、体外细胞存活率检测
(1)实验材料
所用的实验材料大体与实施例6步骤1相同，区别仅在于：本实例使用双去甲氧基姜黄素(BDCur，购于Sigma公司)取代实施例6的姜黄素。
(2)实验方法
操作大体与实施例7步骤1相同，区别仅在于：本实例使用双去甲氧基姜黄素取代实施例7的去甲氧基姜黄素。
(3)实验结果
如图13所示，当先加入20μg/ml NB处理12h再加40μM BDCur处理24h之后对肝癌细胞毒性最大。当双去甲氧基姜黄素(BDCur)浓度为40μM时，其细胞存活率为78.98％，然而当先加入20μg/ml NB处理12h再加40μM BDCur处理24h之后，其细胞存活率从78.98％下降到55.41％，下降了23.57％。表明，添 加NB之后，能显著提高双去甲氧基姜黄素对HepG2细胞的细胞毒性。
2、流式细胞分析
(1)实验材料
PI染料
(2)实验方法
操作大体与实施例7步骤2相同，区别仅在于：本实例使用双去甲氧基姜黄素取代实施例7的去甲氧基姜黄素。
(3)结果与分析
如图14所示，相比对照组而言，NB组内并没有出现明显的凋亡细胞峰，其SubG-1仅为0.6％，且G2/M期细胞数目为12％；BDCur组处理后凋亡细胞峰为2.3％，且G2/M期细胞数目为17.5％，相比BDCur组，NB+BDCur组中的细胞凋亡峰并没有显著提高，仅为1.5％，而G2/M期细胞从17.5％提高到29.5％，表明加入NB后，能显著提高BDCur诱导细胞内的G2/M期细胞阻滞，而SubG1峰的数目并没有显著增加，表明NB和BDCur并不是通过诱导细胞凋亡来抑制细胞增殖。
三、ROS活性检测
(1)实验材料
DHE荧光探针
(2)实验方法
操作大体与实施例6步骤3相同，区别仅在于：本实例使用双去甲氧基姜黄素取代实施例6的姜黄素。
(3)结果与分析
如图15所示，相比对照组来说，NB组的细胞内ROS基本无明显趋势，BDCur组的细胞内ROS升高，而相比BDCur组而言，NB和BDCur联合处理组其ROS进一步提高，并呈时间依赖性。实验结果表明，NB能显著提高BDCur诱导细胞内ROS的产生。
上述实施例为本发明较佳的实施方式，但本发明的实施方式并不受上述实施例的限制，其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化，均应为等效的置换方式，都包含在本发明的保护范围之内。

Claims (10)

  1. 右旋龙脑作为抗肿瘤药物增敏剂的应用。
  2. 根据权利要求1所述的右旋龙脑作为抗肿瘤药物增敏剂的应用，其特征在于：所述的抗肿瘤药物为抗肿瘤化学药物。
  3. 根据权利要求2所述的右旋龙脑作为抗肿瘤药物增敏剂的应用，其特征在于：所述的抗肿瘤化学药物为柔红霉素、阿霉素、去甲氧柔红霉素、表阿霉素、紫杉醇、香菇多糖、长春花碱、长春新碱、三苯氧胺、福美司坦、阿那曲唑、氟他胺、5-氟尿嘧啶、甲氨蝶呤、顺铂、卡铂、奥沙利铂、卡莫司汀、托瑞米芬、替加氟、姜黄素、去甲氧基姜黄素、双去甲氧基姜黄素和塞替派中的至少一种。
  4. 根据权利要求1所述的右旋龙脑作为抗肿瘤药物增敏剂的应用，其特征在于：所述的肿瘤为肝癌、肺癌、恶性黑色瘤、乳腺癌、结肠癌、鼻癌、膀胱癌、宫颈癌、胃癌、食管癌或前列腺癌。
  5. 一种抗肿瘤药物增敏剂，其特征在于：由右旋龙脑和药物允许的赋形剂或载体组成。
  6. 一种抗肿瘤药物，其特征在于：由权利要求5所述的肿瘤药物增敏剂和抗肿瘤药物组成，成分组成为右旋龙脑、赋形剂或载体、和抗肿瘤药物。
  7. 根据权利要求6所述的抗肿瘤药物，其特征在于：所述的抗肿瘤药物为抗肿瘤化学药物。
  8. 根据权利要求6所述的抗肿瘤药物，其特征在于：所述的抗肿瘤化学药物为柔红霉素、阿霉素、去甲氧柔红霉素、表阿霉素、紫杉醇、香菇多糖、长春花碱、长春新碱、三苯氧胺、福美司坦、阿那曲唑、氟他胺、5-氟尿嘧啶、甲氨蝶呤、顺铂、卡铂、奥沙利铂、卡莫司汀、托瑞米芬、替加氟、姜黄素、去甲氧基姜黄素、双去甲氧基姜黄素和塞替派中的至少一种。
  9. 根据权利要求8所述的抗肿瘤药物，其特征在于：所述的抗肿瘤药物由右旋龙脑、赋形剂或载体、和姜黄素组成；所述的姜黄素与所述的右旋龙脑按质量比73674～294696：400000配比。
  10. 根据权利要求8所述的抗肿瘤药物，其特征在于：所述的抗肿瘤药物由右旋龙脑、赋形剂或载体、和姜黄素类化合物组成；所述的右旋龙脑和所述的姜黄素类化合物按质量摩尔比2～8μg：2～8nmol配比。
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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101193682A (zh) * 2005-05-13 2008-06-04 先进科学发展公司 包含一种抗病毒、一种抗肿瘤或一种抗寄生虫药以及一种选自香芹醇、麝香草酚、丁香酚、樟醇和香芹酚的活性物质的药物组合物
CN105147647A (zh) * 2015-08-19 2015-12-16 华南理工大学 右旋龙脑作为抗肿瘤药物增敏剂的应用

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101193682A (zh) * 2005-05-13 2008-06-04 先进科学发展公司 包含一种抗病毒、一种抗肿瘤或一种抗寄生虫药以及一种选自香芹醇、麝香草酚、丁香酚、樟醇和香芹酚的活性物质的药物组合物
CN105147647A (zh) * 2015-08-19 2015-12-16 华南理工大学 右旋龙脑作为抗肿瘤药物增敏剂的应用

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
CHEN, JIANPING: "Molecular mechanism underlying natural borneol potentiated curcuminoids inhibiting HepG2 human hepatocellular carcinoma cells growth", CNKI CHINESE DOCTORAL DISSERTATION DATABASE, 6 June 2015 (2015-06-06) *
LI, LIN ET AL.: "Molecular mechanism underlying natural borneol enhanced curcuminoids inducing melanoma cell apoptosis", PROCEEDINGS OF THE ACADEMIC SYMPOSIUM OF BIOPHYSICS SOCIETY OF GUANGDONG PROVINCE, 30 September 2014 (2014-09-30) *
ZHENG, XIAOWEI: "Effect of borneol on reverse the multidrug resistance in cancer", SICHUAN JOURNAL OF PHYSIOLOGICAL SCIENCE, vol. 29, no. 3, 31 December 2007 (2007-12-31) *

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