广西何首乌蒽醌化合物的分离提取及GXHSWAQ-1对鼻咽癌放射增敏作用的研究
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摘要
鼻咽癌(nasopharyngeal carcinoma,NPC)是广西常见的恶性肿瘤之一,年发病率可高至30～50/10万。由于鼻咽部解剖位置的特殊性及鼻咽癌细胞对射线较为敏感的特点,放射治疗成为鼻咽癌的首选治疗,但放射治疗的结果往往未达到人们所期望的效果。数据表明,放射结束时,10%～18%的患者有局部病灶残留,20%～30%的患者有局部和颈部复发,20%～36%的患者发生远处转移。究其失败原因是肿瘤细胞对射线的抗拒性,因此增强肿瘤细胞对射线的敏感性,是提高放疗效果的重要途径。
实验证明,中药放射增敏剂——蒽醌类和泛醌类化合物,是一类具有放射增敏作用且毒性较低的化合物。目前人们对其放射增敏机制尚缺乏了解,而药物作用后细胞DNA双链损伤和修复相关基因、蛋白质发生怎样的改变,亦无文献报道。广西何首乌为一味传统中药,是蓼科植物的干燥块根,富含葸醌类、二苯乙烯苷类、磷脂类等成分。若采用现代色谱分离技术,从广西特有的植物何首乌中,筛选出结构新颖、疗效显著、毒性低、价格低廉的天然放疗增敏剂——蒽醌类化合物,对于充分利用广西资源,加快中药现代化进程,提高肿瘤放射治疗的效益,具有重要而深远的意义。
本课题采用不同的溶剂对广西中药材何首乌醇提物进行分步萃取,并对乙酸乙酯层经多次硅胶柱层析、重结晶纯化等步骤,分离获得七个化合物,分别测定了它们的IR、~1HNMR和部分化合物的~(18)CNMR图谱。经化学结构解析,它们分别是:大黄素甲醚(化合物Ⅰ)、大黄素(化合物Ⅱ)、大黄素-8-甲醚(化合物Ⅲ)、β-谷甾醇(化合物Ⅳ)、没食子酸(化合物Ⅴ)、胡萝卜苷(化合物Ⅵ)、GXHSWAQ-1(Ⅶ)。为了探讨蒽醌类化合物GXHSWAQ-1对放射剂量为2Gy不敏感的鼻咽癌CNE-1细胞放射增敏作用及对放射增敏相关基因的影响。通过MTT比色法检测GXHSWAQ-1对鼻咽癌CNE-1细胞株的生长抑制作用以及确定其在体外增敏剂量和放射增敏比,逆转录-聚合酶链反应(RT-PCR)检测GXHSWAQ-1作用前后,细胞放射敏感性基因Ku70、Ku80、HIF-1及ATM的表达情况。结果发现:GXHSWAQ-1在浓度低于62.5μg/ml时对鼻咽癌CNE-1细胞无生长抑制作用(抑制率低于10%),采用7.8μg/ml浓度时能抑制照射后CNE-1细胞的生长,其放射增敏比高于安卡胶囊,对放射敏感相关基因Ku70、Ku80、HIF-1及ATM的表达均有下调的作用。GXHSWAQ-1对鼻咽癌CNE-1细胞具有放射增敏作用,其作用机制与Ku70、Ku80、ATM、HIF-1相关。
Nasopharyngeal carcinoma(NPC)is one of the common malignant tumors in Guangxi province,China.The incidence of NPC is as high as 30-50 per 100000 individuals.Owing to its special anatomic site and characteristic of radiosensitivity,the primary treating method lies in radiotherapy.But the radiotherapeutic treatment is not desirable.The data indicat that,about 10%to 18%NPC patients who had residual focus,20%to 30%NPC sufferers who had local recurrence,20%to 36%NPC sufferers who had distanct metastasis,one of the major causes of treatment failure was radioresisitant.Hence,to enhance the sensitivity of NPC cells to radiation is one way to improve the therapeutic effects.
Anthraquinone and Ubiquinone compounds from Chinese herbs medicine are radiosensitizing agents,but the mechanism is not clear.Polygonum multiflorum thumb.,the dried roots of Polygonaceae botanic,is one of the common chinese traditional medicines in Gunagxi province.Its chemical constituents contain anthraquinones,diphenyl ethylenes and phospholipins.If we separated natural compounds from Polygonum multiflorum thumb,by modern chromatographic separation techniques,we would obtain some bioactive compounds and drug candidates which are cheaper,lower cytotoxicity and more effective therapeutic power.It is important and profound meaning for utilizing the natural resources of Guangxi province,accelerating development of the Modernization of Traditional Chinese Medicine,and improving the effectiveness of radiotherapy.
7 compounds were isolated and purified from ethyl acetate extraction of Polygonum multiflorum thumb,by extraction with different organic solvents, various column chromatography,and recrystallisation methods.Spectroscopic data including IR,~1HNMR,and ~(13)CNMR of those compounds were established for their chemical structural elucidateon.As a result,7 compounds were identified as:physcion(Ⅰ),emodin(Ⅱ),questin(Ⅲ),β-sitosterol(Ⅳ),gallic acid(Ⅴ),daucosterol(Ⅵ),GXHSWAQ-1(Ⅶ).To investigate the radiosensitization of GXHSWAQ-1(an Anthraquinone compound extracted from Polygonum multiflorum thumb.)in human nasopharyngeal carcinoma cell lines CNE-1 which is radioresistance tumor cells for 2 Gy and the expression of Ku70, Ku80,HIF-1 and ATM after GXHSWAQ-1 treated.The inhibition effects of GXHSWAQ-1 and sensitive enhancement ratio(SER)on thehuman CNE-1 cell lines were evaluated by MTT colorimetric assay.Ku70,Ku80,HIF-1 and ATM genes expression were detected by RT-PCR.The result showed that when the concentration of GXHSWAQ-1 was lower than 62.5μg/ml,there is no cytotoxicity for CNE-1 cell lines(Inhibitory ratio is lower than 10%).But at the concentration of 7.8μg/ml GXHSWAQ-1 could inhibited cells growth post-irradiation.The sensitive enhancement ratio(SER)of GXHSWAQ-1 was higher than that of Anka(Irisquinone capsules).The radio sensitivity related gene Ku70,Ku80,HIF-1 and ATM in CNE-1 cells were down-regulationafter treated with different concentration of GXHSWAQ-1.The result show that GXHSWAQ-1 is radiosensitizing effect in vitro on Nasopharyngeal Carcinoma cells.Radiosensitization by GXHSWAQ-1 is possibly associated with the expression ofku80,ku70,HIF-1 and ATM.
实验证明,中药放射增敏剂——蒽醌类和泛醌类化合物,是一类具有放射增敏作用且毒性较低的化合物。目前人们对其放射增敏机制尚缺乏了解,而药物作用后细胞DNA双链损伤和修复相关基因、蛋白质发生怎样的改变,亦无文献报道。广西何首乌为一味传统中药,是蓼科植物的干燥块根,富含葸醌类、二苯乙烯苷类、磷脂类等成分。若采用现代色谱分离技术,从广西特有的植物何首乌中,筛选出结构新颖、疗效显著、毒性低、价格低廉的天然放疗增敏剂——蒽醌类化合物,对于充分利用广西资源,加快中药现代化进程,提高肿瘤放射治疗的效益,具有重要而深远的意义。
本课题采用不同的溶剂对广西中药材何首乌醇提物进行分步萃取,并对乙酸乙酯层经多次硅胶柱层析、重结晶纯化等步骤,分离获得七个化合物,分别测定了它们的IR、~1HNMR和部分化合物的~(18)CNMR图谱。经化学结构解析,它们分别是:大黄素甲醚(化合物Ⅰ)、大黄素(化合物Ⅱ)、大黄素-8-甲醚(化合物Ⅲ)、β-谷甾醇(化合物Ⅳ)、没食子酸(化合物Ⅴ)、胡萝卜苷(化合物Ⅵ)、GXHSWAQ-1(Ⅶ)。为了探讨蒽醌类化合物GXHSWAQ-1对放射剂量为2Gy不敏感的鼻咽癌CNE-1细胞放射增敏作用及对放射增敏相关基因的影响。通过MTT比色法检测GXHSWAQ-1对鼻咽癌CNE-1细胞株的生长抑制作用以及确定其在体外增敏剂量和放射增敏比,逆转录-聚合酶链反应(RT-PCR)检测GXHSWAQ-1作用前后,细胞放射敏感性基因Ku70、Ku80、HIF-1及ATM的表达情况。结果发现:GXHSWAQ-1在浓度低于62.5μg/ml时对鼻咽癌CNE-1细胞无生长抑制作用(抑制率低于10%),采用7.8μg/ml浓度时能抑制照射后CNE-1细胞的生长,其放射增敏比高于安卡胶囊,对放射敏感相关基因Ku70、Ku80、HIF-1及ATM的表达均有下调的作用。GXHSWAQ-1对鼻咽癌CNE-1细胞具有放射增敏作用,其作用机制与Ku70、Ku80、ATM、HIF-1相关。
Nasopharyngeal carcinoma(NPC)is one of the common malignant tumors in Guangxi province,China.The incidence of NPC is as high as 30-50 per 100000 individuals.Owing to its special anatomic site and characteristic of radiosensitivity,the primary treating method lies in radiotherapy.But the radiotherapeutic treatment is not desirable.The data indicat that,about 10%to 18%NPC patients who had residual focus,20%to 30%NPC sufferers who had local recurrence,20%to 36%NPC sufferers who had distanct metastasis,one of the major causes of treatment failure was radioresisitant.Hence,to enhance the sensitivity of NPC cells to radiation is one way to improve the therapeutic effects.
Anthraquinone and Ubiquinone compounds from Chinese herbs medicine are radiosensitizing agents,but the mechanism is not clear.Polygonum multiflorum thumb.,the dried roots of Polygonaceae botanic,is one of the common chinese traditional medicines in Gunagxi province.Its chemical constituents contain anthraquinones,diphenyl ethylenes and phospholipins.If we separated natural compounds from Polygonum multiflorum thumb,by modern chromatographic separation techniques,we would obtain some bioactive compounds and drug candidates which are cheaper,lower cytotoxicity and more effective therapeutic power.It is important and profound meaning for utilizing the natural resources of Guangxi province,accelerating development of the Modernization of Traditional Chinese Medicine,and improving the effectiveness of radiotherapy.
7 compounds were isolated and purified from ethyl acetate extraction of Polygonum multiflorum thumb,by extraction with different organic solvents, various column chromatography,and recrystallisation methods.Spectroscopic data including IR,~1HNMR,and ~(13)CNMR of those compounds were established for their chemical structural elucidateon.As a result,7 compounds were identified as:physcion(Ⅰ),emodin(Ⅱ),questin(Ⅲ),β-sitosterol(Ⅳ),gallic acid(Ⅴ),daucosterol(Ⅵ),GXHSWAQ-1(Ⅶ).To investigate the radiosensitization of GXHSWAQ-1(an Anthraquinone compound extracted from Polygonum multiflorum thumb.)in human nasopharyngeal carcinoma cell lines CNE-1 which is radioresistance tumor cells for 2 Gy and the expression of Ku70, Ku80,HIF-1 and ATM after GXHSWAQ-1 treated.The inhibition effects of GXHSWAQ-1 and sensitive enhancement ratio(SER)on thehuman CNE-1 cell lines were evaluated by MTT colorimetric assay.Ku70,Ku80,HIF-1 and ATM genes expression were detected by RT-PCR.The result showed that when the concentration of GXHSWAQ-1 was lower than 62.5μg/ml,there is no cytotoxicity for CNE-1 cell lines(Inhibitory ratio is lower than 10%).But at the concentration of 7.8μg/ml GXHSWAQ-1 could inhibited cells growth post-irradiation.The sensitive enhancement ratio(SER)of GXHSWAQ-1 was higher than that of Anka(Irisquinone capsules).The radio sensitivity related gene Ku70,Ku80,HIF-1 and ATM in CNE-1 cells were down-regulationafter treated with different concentration of GXHSWAQ-1.The result show that GXHSWAQ-1 is radiosensitizing effect in vitro on Nasopharyngeal Carcinoma cells.Radiosensitization by GXHSWAQ-1 is possibly associated with the expression ofku80,ku70,HIF-1 and ATM.
引文
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