CN112375803A - 五倍子中抗炎活性成分没食子酸乙酯的筛选方法及其用途 - Google Patents
五倍子中抗炎活性成分没食子酸乙酯的筛选方法及其用途 Download PDFInfo
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- CN112375803A CN112375803A CN202011278964.4A CN202011278964A CN112375803A CN 112375803 A CN112375803 A CN 112375803A CN 202011278964 A CN202011278964 A CN 202011278964A CN 112375803 A CN112375803 A CN 112375803A
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- Prior art keywords
- ethyl gallate
- galloyl glucose
- gallnut
- cervicitis
- galloyl
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Abstract
本发明涉及五倍子中抗炎活性成分没食子酸乙酯的筛选方法及其用途,该方法利用液质联用纯化分离系统分离五倍子中各成分并通过体内外实验筛选五倍子中治疗宫颈炎的活性成分。体内药理研究表明:没食子酸乙酯对宫颈炎大鼠有明显的治疗作用。没食子酸乙酯可以改善宫颈炎大鼠阴道红肿的情况,减少宫颈分泌物,抑制宫颈脏器指数的增加;病理组织切片结果显示没食子酸乙酯可减轻炎性细胞的浸润,加速修复宫颈粘膜上皮;酶联免疫吸附(ELISA)试验表明没食子酸乙酯可以抑制宫颈炎大鼠肿瘤坏死因子‑α(TNF‑α)的表达,促进分泌表皮生长因子(EGF)和表皮生长因子受体(EGFR)。
Description
技术领域
本发明属于医药领域,涉及五倍子中抗炎活性成分没食子酸乙酯的筛选方法及其用途。
背景技术
宫颈炎和非特异性宫颈炎的研究属于疾病病因不明的性传播感染(sexuallytransmitted infection,STI)医学研究领域。近年来,宫颈炎的发病率呈持续上升的趋势,且患病人群越来越趋于年轻化。由于宫颈管黏膜上皮为单层柱状上皮,抗感染能力较差,易发生感染。宫颈感染从下生殖道(阴道)开始,然后发展为盆腔炎、上生殖道(子宫和输卵管)和腹腔上升感染。临床多见的宫颈炎是急性子宫颈管黏膜炎,若急性子宫颈炎未经及时诊治或病原体持续存在,可导致慢性子宫颈炎症;严重者会导致进一步的不孕和异位妊娠。研究表明,宫颈炎是病原体侵入损伤部位所致,常见病原体分为性传播疾病病原体和内源性病原体。性传播疾病病原体包括淋病奈瑟菌及沙眼衣原体,内源性病原体包括葡萄球菌、链球菌、大肠杆菌以及滴虫、念珠菌、阿米巴原虫等。临床上的治疗药物主要是抗生素,由于抗生素滥用和假阳性诊断导致耐药病原体的出现,该疾病的复发率呈逐渐上升的趋势。
五倍子为漆树科植物盐肤木(Rhus chinensis Mill.)、青麸杨(Rhus potaniniiMaxim.)或红麸杨(Rhus punjabensis Stew.var.sinica(Diels)Rehd.et Wils.)叶上的虫瘿,主要由五倍子蚜寄生而形成。它主要分布于四川、贵州、云南、陕西、广西等地,其主要成分为没食子酸和鞣质,其中没食子鞣质含量高达50%~70%。临床研究发现,五倍子具有止泻、抗菌、抗炎、抗氧化、抗病毒、抗衰老和降血糖等药理作用,此外还有抗癌、抗生育、清除自由基等作用。夏尔热·依布拉合木等人在《维吾尔医治疗300例慢性宫颈炎的临床报告》一文中报道外用复方没食子栓剂治疗慢性宫颈炎;曾佳烽(专利号:CN201210129001.7)等人发现多花野牡丹提取物中包含2%~15%的氨基酸、1%~7%的没食子酸、1%~8%的柠檬酸、2%~10%的蔗糖和0.5%~3.5%的有机铝,通过与载体材料结合制成凝胶剂、栓剂、乳剂、霜剂或洗剂可用于预防和治疗宫颈炎、宫颈糜烂。
天然产物一直是新药研究和开发的宝贵资源。五倍子作为传统中药,从中寻找治疗宫颈炎的活性物质并探究治疗机制,为五倍子的进一步开发利用提供思路,同时也为临床上宫颈炎的诊断治疗提供新策略。没食子酸乙酯是从五倍子中分离出来的一种酚酸类化合物。目前有关筛选五倍子中抗炎活性的成分及没食子酸乙酯在治疗宫颈炎方面的应用未见报道。
发明内容
本发明的目的在于,提供一种五倍子中抗炎活性成分没食子酸乙酯的筛选方法及其用途,该方法利用液质联用纯化分离系统分离五倍子中各成分并通过体内外实验筛选五倍子中治疗宫颈炎的活性成分。体内药理研究表明:没食子酸乙酯对宫颈炎大鼠有明显的治疗作用。没食子酸乙酯可以改善宫颈炎大鼠阴道红肿的情况,减少宫颈分泌物,抑制宫颈脏器指数的增加;病理组织切片结果显示没食子酸乙酯可减轻炎性细胞的浸润,加速修复宫颈粘膜上皮;酶联免疫吸附(ELISA)试验表明没食子酸乙酯可以抑制宫颈炎大鼠肿瘤坏死因子-α(TNF-α)的表达,促进分泌表皮生长因子(EGF)和表皮生长因子受体(EGFR)。
本发明所述的一种五倍子中抗炎活性成分没食子酸乙酯的筛选方法,按下列步骤进行:
a、选用漆树科植物盐肤木、青麸杨或红麸杨叶上的虫瘿作为五倍子药材;
b、将步骤a中五倍子药材粉碎过60目筛,称取100g药材粉末,以300mL蒸馏水为溶剂回流提取3次,每次提取2小时,合并提取液;
c、将步骤b得到的合并提取液,使用X Bridge C18色谱柱对五倍子中的各成分进行纯化,分离得到的五倍子中11种成分包含:没食子酸,一-O-没食子酰基葡萄糖,二-O-没食子酰基葡萄糖,双没食子酸,三-O-没食子酰基葡萄糖,没食子酸乙酯,四-O-没食子酰基葡萄糖,五-O-没食子酰基葡萄糖,六-O-没食子酰基葡萄糖,双没食子酸乙酯和七-O-没食子酰基葡萄糖;
d、通过体外实验筛选五倍子中抗炎活性成分:以脂多糖诱导RAW264.7巨噬细胞形成炎症模型,通过噻唑蓝法测定五倍子中各成分对RAW264.7巨噬细胞生长活力的影响以及通过酶联免疫吸附法检测RAW264.7细胞上清液中一氧化氮、白介素-6、白介素-1β和肿瘤坏死因子-α的表达以及流式细胞术检测RAW264.7细胞中活性氧表达的影响,实验筛选出五倍子中抗炎活性成分为没食子酸乙酯。
所述方法获得的五倍子中抗炎活性成分没食子酸乙酯在制备治疗宫颈炎的药物中的用途。
本发明所述的一种五倍子中抗炎活性成分没食子酸乙酯的筛选方法,该方法将五倍子药材粉碎过筛,回流提取,合并提取液,采用液质联用纯化分离系统分离并得到五倍子中11种成分,包含:没食子酸、一-O-没食子酰基葡萄糖、二-O-没食子酰基葡萄糖、双没食子酸、三-O-没食子酰基葡萄糖、没食子酸乙酯、四-O-没食子酰基葡萄糖、五-O-没食子酰基葡萄糖、六-O-没食子酰基葡萄糖、双没食子酸乙酯和七-O-没食子酰基葡萄糖。通过体外细胞活力实验,各成分对脂多糖(LPS)诱导的RAW264.7细胞中一氧化氮(NO)、白介素-6(IL-6)、白介素-1β(IL-1β)和肿瘤坏死因子-α(TNF-α)表达以及流式细胞术检测RAW264.7细胞中活性氧(ROS)表达的影响。我们发现没食子酸乙酯的安全浓度(细胞存活超过80%的概率)为25μg/mL,而且可以明显减轻由脂多糖(LPS)引起RAW264.7细胞中一氧化氮(NO)、白介素-6(IL-6)、白介素-1β(IL-1β)和肿瘤坏死因子-α(TNF-α)表达增加以及活性氧(ROS)升高的情况。体内药理研究表明:没食子酸乙酯对宫颈炎大鼠有明显的治疗作用。没食子酸乙酯可以改善宫颈炎大鼠阴道红肿的情况,减少宫颈分泌物,抑制宫颈脏器指数的增加;病理组织切片结果显示没食子酸乙酯可减轻炎性细胞的浸润,加速修复宫颈粘膜上皮;酶联免疫吸附(ELISA)试验表明没食子酸乙酯可以抑制宫颈炎大鼠肿瘤坏死因子-α(TNF-α)的表达,促进分泌表皮生长因子(EGF)和表皮生长因子受体(EGFR)。
本发明本发明所述的一种五倍子中抗炎活性成分没食子酸乙酯的筛选方法,该方法中选用的是漆树科植物盐肤木(Rhus chinensis Mill.)、青麸杨(Rhus potaniniiMaxim.)或红麸杨(Rhus punjabensis Stew.var.sinica(Diels)Rehd.et Wils.)叶上的虫瘿作为五倍子药材;
将五倍子药材粉碎过60目筛,称取100g药材粉末,以300mL蒸馏水为溶剂回流提取3次,每次提取2小时,合并提取液;
将得到的合并提取液,使用X Bridge C18色谱柱对五倍子中的各成分进行纯化制备。分离得到的五倍子中11种成分包含:没食子酸,一-O-没食子酰基葡萄糖,二-O-没食子酰基葡萄糖,双没食子酸,三-O-没食子酰基葡萄糖,没食子酸乙酯,四-O-没食子酰基葡萄糖,五-O-没食子酰基葡萄糖,六-O-没食子酰基葡萄糖,双没食子酸乙酯和七-O-没食子酰基葡萄糖;首先通过体外实验筛选五倍子中抗炎活性成分:以脂多糖(LPS)诱导RAW264.7巨噬细胞形成炎症模型,通过噻唑蓝(MTT)法测定五倍子中各成分对RAW264.7巨噬细胞生长活力的影响以及通过酶联免疫吸附(ELISA)法检测RAW264.7细胞上清液中一氧化氮(NO)、白介素-6(IL-6)、白介素-1β(IL-1β)和肿瘤坏死因子-α(TNF-α)的表达以及流式细胞术检测RAW264.7细胞中活性氧(ROS)表达的影响。结果表明:噻唑蓝(MTT)试验表明一-O-没食子酰基葡萄糖、二-O-没食子酰基葡萄糖、双没食子酸、三-O-没食子酰基葡萄糖、没食子酸乙酯、四-O-没食子酰基葡萄糖的安全浓度高(细胞存活超过80%的概率);酶联免疫吸附(ELISA)试验表明二-O-没食子酰基葡萄糖、三-O-没食子酰基葡萄糖和没食子酸乙酯降低一氧化氮(NO)、白介素-6(IL-6)、白介素-1β(IL-1β)和肿瘤坏死因子-α(TNF-α)的表达明显;流式细胞术检测表明没食子酸乙酯明显降低细胞内活性氧(ROS)产生。通过体外实验筛选五倍子中抗炎活性成分为没食子酸乙酯。
进一步研究没食子酸乙酯对宫颈炎大鼠的治疗作用以25%苯酚胶浆诱导建立宫颈炎模型,造模成功后用没食子酸乙酯治疗进行评价。对大鼠外阴红肿和阴道分泌物进行评分、检测宫颈重量及脏器指数、检测大鼠血清中的瘤坏死因子α(TNF-α)、表皮生长因子(EGF)和表皮生长因子受体(EGFR)的表达情况,并对宫颈组织进行病理学切片。试验结果发现,给予25%苯酚胶浆的大鼠阴道口红肿,有粘性分泌物流出,毛色变差,食欲下降;与模型组相比,给予药物治疗后,阳性药组、没食子酸乙酯组的大鼠逐渐恢复了行动和饮食,且大鼠阴道红肿和阴道分泌物的情况逐渐好转,红肿逐渐消失,阴道分泌物逐渐减少甚至消失。没食子酸乙酯可以抑制宫颈脏器指数的增加。病理组织切片表明宫颈组织上皮厚度增加,角化层脱落,粘膜及其固有层充血水肿,较多炎细胞浸润,腔内有大量炎性渗出物及坏死组织;与模型组相比,阳性药组、没食子酸乙酯组的宫颈组织病变减轻,病变处的部分组织已经开始得到修复,炎性细胞的浸润减轻,炎性渗出减少。经给药治疗后,较模型组来说,治疗组大鼠的肿瘤坏死因子-α(TNF-α)水平显著降低(P<0.001);没食子酸乙酯治疗显著增加表皮生长因子(EGF)和表皮生长因子受体(EGFR)的表达(P<0.001)。
附图说明
图1为本发明五倍子水提液及液质联用纯化系统分离得到的五倍子各成分液相色谱图,其中A为七-O-没食子酰基葡萄糖液相色谱图,B为双没食子酸乙酯液相色谱图,C为六-O-没食子酰基葡萄糖液相色谱图,D为五-O-没食子酰基葡萄糖液相色谱图,E为四-O-没食子酰基葡萄糖液相色谱图,F为没食子酸乙酯液相色谱图,G为三-O-没食子酰基葡萄糖液相色谱图,H为双没食子酸液相色谱图,I为二-O-没食子酰基葡萄糖液相色谱图,J为一-O-没食子酰基葡萄糖液相色谱图,K为没食子酸液相色谱图,L为五倍子水提液液相色谱图。
图2为本发明噻唑蓝(MTT)法评价五倍子各成分对RAW264.7细胞生长活力影响。数据表示为平均值±S.D,#与空白组相比,#P<0.05,##P<0.01,###P<0.001;*与模型组相比,*P<0.05,**P<0.01,***P<0.001。a是阳性药地塞米松,b是没食子酸,c是双没食子酸,d是一-O-没食子酰基葡萄糖,e是二-O-没食子酰基葡萄糖,f是三-O-没食子酰基葡萄糖,g是没食子酸乙酯,h是四-O-没食子酰基葡萄糖,i是双没食子酸乙酯,j是五-O-没食子酰基葡萄糖,k是六-O-没食子酰基葡萄糖,l是七-O-没食子酰基葡萄糖。
图3为本发明酶联免疫吸附(ELISA)法检测双没食子酸、一-O-没食子酰基葡萄糖、二-O-没食子酰基葡萄糖、三-O-没食子酰基葡萄糖、没食子酸乙酯、四-O-没食子酰基葡萄糖对RAW264.7细胞内一氧化氮(NO)的影响。数据表示为平均值±S.D,#与空白组相比,#P<0.05,##P<0.01,###P<0.001;*与模型组相比,*P<0.05,**P<0.01,***P<0.001。a是阳性药地塞米松,c是双没食子酸,d是一-O-没食子酰基葡萄糖,e是二-O-没食子酰基葡萄糖,f是三-O-没食子酰基葡萄糖,g是没食子酸乙酯,h是四-O-没食子酰基葡萄糖。
图4为本发明酶联免疫吸附(ELISA)法检测双没食子酸、一-O-没食子酰基葡萄糖、二-O-没食子酰基葡萄糖、三-O-没食子酰基葡萄糖、没食子酸乙酯、四-O-没食子酰基葡萄糖对RAW264.7细胞内白介素-6(IL-6)表达的影响。数据表示为平均值±S.D,#与空白组相比,#P<0.05,##P<0.01,###P<0.001;*与模型组相比,*P<0.05,**P<0.01,***P<0.001。a是阳性药地塞米松,c是双没食子酸,d是一-O-没食子酰基葡萄糖,e是二-O-没食子酰基葡萄糖,f是三-O-没食子酰基葡萄糖,g是没食子酸乙酯,h是四-O-没食子酰基葡萄糖。
图5为本发明酶联免疫吸附(ELISA)法检测双没食子酸、一-O-没食子酰基葡萄糖、二-O-没食子酰基葡萄糖、三-O-没食子酰基葡萄糖、没食子酸乙酯、四-O-没食子酰基葡萄糖对RAW264.7细胞内白介素-1β(IL-1β)表达的影响。数据表示为平均值±S.D,#与空白组相比,#P<0.05,##P<0.01,###P<0.001;*与模型组相比,*P<0.05,**P<0.01,***P<0.001。a是阳性药地塞米松,c是双没食子酸,d是一-O-没食子酰基葡萄糖,e是二-O-没食子酰基葡萄糖,f是三-O-没食子酰基葡萄糖,g是没食子酸乙酯,h是四-O-没食子酰基葡萄糖。
图6为本发明酶联免疫吸附(ELISA)法检测双没食子酸、一-O-没食子酰基葡萄糖、二-O-没食子酰基葡萄糖、三-O-没食子酰基葡萄糖、没食子酸乙酯、四-O-没食子酰基葡萄糖对RAW264.7细胞内肿瘤坏死因子-α(TNF-α)表达的影响。数据表示为平均值±S.D,#与空白组相比,#P<0.05,##P<0.01,###P<0.001;*与模型组相比,*P<0.05,**P<0.01,***P<0.001,a是阳性药地塞米松,c是双没食子酸,d是一-O-没食子酰基葡萄糖,e是二-O-没食子酰基葡萄糖,f是三-O-没食子酰基葡萄糖,g是没食子酸乙酯,h是四-O-没食子酰基葡萄糖。
图7为本发明流式细胞术检测二-O-没食子酰基葡萄糖、三-O-没食子酰基葡萄糖和没食子酸乙酯对RAW264.7细胞内活性氧(ROS)产生的影响(A)以及数据统计结果(B)。数据表示为平均值±S.D,#与空白组相比,#P<0.05,##P<0.01,###P<0.001;*与模型组相比,*P<0.05,**P<0.01,***P<0.001。e是二-O-没食子酰基葡萄糖,f是三-O-没食子酰基葡萄糖,g是没食子酸乙酯。
图8为本发明没食子酸乙酯治疗宫颈炎大鼠的阴道红肿评分(A)、宫颈分泌物评分(B)和脏器指数(C)。数据表示为平均值±S.D,#与空白组相比,#P<0.05,##P<0.01,###P<0.001;*与模型组相比,*P<0.05,**P<0.01,***P<0.001。a是空白组,b是模型组,c是阳性药组,d是没食子酸乙酯给药组。
图9为本发明宫颈的组织病理学切片(A)及组织病理学评分(B)。数据表示为平均值±S.D,#与空白组相比,#P<0.05,##P<0.01,###P<0.001;*与模型组相比,*P<0.05,**P<0.01,***P<0.001。a是空白组,b是模型组,c是阳性药组,d是没食子酸乙酯给药组。
图10为本发明没食子酸乙酯对大鼠宫颈组织肿瘤坏死因子-α(TNF-α)(A)、表皮生长因子(EGF)(B)和表皮生长因子受体(EGFR)(C)的影响。数据表示为平均值±S.D,#与空白组相比,#P<0.05,##P<0.01,###P<0.001;*与模型组相比,*P<0.05,**P<0.01,***P<0.001。a是空白组,b是模型组,c是阳性药组,d是没食子酸乙酯给药组。
具体实施方式
实施例1
液质联用纯化系统分离五倍子各成分:
a、选用的是漆树科植物盐肤木(Rhus chinensis Mill.)、青麸杨(Rhuspotaninii Maxim.)或红麸杨(Rhus punjabensis Stew.var.sinica(Diels)Rehd.etWils.)叶上的虫瘿作为五倍子药材;
b、将五倍子药材粉碎过60目筛,称取100g药材粉末,以300mL蒸馏水为溶剂回流提取3次,每次提取2小时,合并提取液;
c、将步骤b得到的合并提取液,使用X Bridge C18色谱柱对五倍子中的各成分进行纯化制备,以17mL/min流速洗脱样品,注射体积20μL,样品浓度1.5mg/mL,以A为0.2%甲酸水溶液,B为乙腈为流动相;洗脱程序为:0–8min为93%–80%A;8–35min为93%–80%A;35–50min为80%–70%A;50–60min为70%–0%A;
光谱条件:254nm和269nm;
ESI-MS条件:电喷雾负离子模式;
扫描范围:100-1000(m/z);
源温度:120℃;去溶剂化温度:250℃;毛细管电压2.8kV;锥孔电压50V;
分离得到的五倍子中11种成分包含:没食子酸,一-O-没食子酰基葡萄糖,二-O-没食子酰基葡萄糖,双没食子酸,三-O-没食子酰基葡萄糖,没食子酸乙酯,四-O-没食子酰基葡萄糖,五-O-没食子酰基葡萄糖,六-O-没食子酰基葡萄糖,双没食子酸乙酯和七-O-没食子酰基葡萄糖;
体外筛选五倍子中抗炎活性成分:
目的:根据体外实验筛选出五倍子中抗炎活性好、毒性低的成分;
方法:通过噻唑蓝(MTT)法测定五倍子中各成分对RAW264.7巨噬细胞生长活力的影响以及通过酶联免疫吸附(ELISA)法检测RAW264.7细胞上清液中一氧化氮(NO)、白介素-6(IL-6)、白介素-1β(IL-1β)和肿瘤坏死因子-α(TNF-α)的表达以及流式细胞术检测RAW264.7细胞中活性氧(ROS)表达的影响;结果:噻唑蓝(MTT)试验表明一-O-没食子酰基葡萄糖、二-O-没食子酰基葡萄糖、双没食子酸、三-O-没食子酰基葡萄糖、没食子酸乙酯、四-O-没食子酰基葡萄糖的安全浓度高,细胞存活超过80%的概率;酶联免疫吸附(ELISA)试验表明二-O-没食子酰基葡萄糖、三-O-没食子酰基葡萄糖和没食子酸乙酯降低一氧化氮(NO)、白介素-6(IL-6)、白介素-1β(IL-1β)和肿瘤坏死因子-α(TNF-α)的表达明显;流式细胞术检测表明没食子酸乙酯明显降低细胞内活性氧(ROS)产生;结论:筛选出五倍子中抗炎活性成分为没食子酸乙酯。
实施例2
实验材料与方法
药物与试剂
五倍子药材购买于陕西汉中,由韩博教授(石河子大学药学院)鉴定为五倍子(Rhus chinensis Mill.);乙腈(色谱级,Fisher Scientific);甲酸(优级,天津光复精细化工研究所);达尔伯克氏改良伊格尔培养基(DMEM,上海Gibco公司);胎儿牛血清(FBS,Biological Industries);PBS缓冲液(Solarbio);脂多糖(LPS,Sigma Chemical);3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四唑鎓溴化物(MTT,Solarbio);二甲基亚砜(DMSO,Solarbio);青霉素-链霉素溶液(Solarbio);胰蛋白酶-EDTA消化液(T1320,北京索莱宝科技有限公司);活性氧测定试剂盒(E004,南京建成生物工程研究所);一氧化氮酶联免疫试剂盒(Cat.#m1003096-C,上海酶联生物科技有限公司);肿瘤坏死因子-α酶联免疫试剂盒(Cat.#m1002095-C,上海酶联生物科技有限公司);白介素-6酶联免疫试剂盒(Cat.#m1002293-C,上海酶联生物科技有限公司);白介素-1β酶联免疫试剂盒(Cat.#m1063132-C,上海酶联生物科技有限公司);大鼠表皮生长因子受体(EGFR)检测试剂盒(上海酶联生物科技有限公司);大鼠表皮生长因子(EGF)检测试剂盒(上海酶联生物科技有限公司);苯酚(上海源叶生物科技有限公司);阿拉伯树胶粉(上海源叶生物科技有限公司);消糜栓(通化万通药业股份有限公司);小鼠巨噬细胞RAW264.7(中国科学院上海细胞库);
噻唑蓝(MTT)法检测五倍子各成分对RAW264.7细胞生长活力的影响:
将对数生长期的RAW264.7细胞以5×104个细胞/孔接种到96孔板中,向各孔中加入100μL的细胞悬浮液,各组提供平行复孔4个。分别设置空白组、脂多糖(LPS)、脂多糖(LPS)与不同浓度成分给药组(3.125、6.25、12.5、25μg/mL)。在37℃,5%CO2的培养箱中培养24h,弃去旧培养基,空白组给予新鲜培养基,给药组按浓度给药,预处理1h后加入脂多糖(LPS)(0.5μg/mL)在培养箱中培养24h。在24h后,避免光照条件下向每个孔中加入20μL MTT溶液(5mg/mL),在37℃培养箱中培养4h,弃去细胞上清溶液,向每孔中加入150μL的DMSO溶解反应后的结晶产物,水平摇床上振摇5~10min使充分混合,在490nm波长处检测各孔的OD值,按公式计算细胞存活率(如式1所示):
细胞存活率%=实验组平均OD值/对照组平均OD值×100% (式1);
酶联免疫吸附(ELISA)法检测一氧化氮(NO)及炎症因子:
将RAW264.7细胞以4.5×105个细胞/孔接种到12孔板,每孔加入细胞悬液1mL,每组设平行复孔2个,设置空白组、脂多糖(LPS)和脂多糖(LPS)与不同浓度成分给药组(3.125、6.25、12.5、25μg/mL),在温度37℃,5%CO2的培养箱中培养24h,弃去旧培养基,空白组给予新鲜培养基,其他组按浓度给药,给药预处理1h后加入脂多糖(LPS)(0.5μg/mL),继续培养24h。收集各组细胞上清液,离心(5000rpm,20min)后分装,于-80℃保存备用。流式细胞术检测细胞内ROS水平:
将RAW264.7细胞以4.5×105个细胞/孔的密度接种在6孔板中,置于37℃,5%CO2培养箱中培养24h。次日弃去旧培养基,设置空白、脂多糖(LPS)和脂多糖(LPS)与不同浓度成分给药组(6.25、12.5、25μg/mL)。培养24h后除空白孔以外,每孔加20μL DCFH-DA探针,并在37℃,5%CO2的培养箱中孵育40min,弃去上清液,每孔加1mL胰酶消化2min,每孔加1mL培养基终止消化;重悬细胞,在1000rpm离心10min,用PBS洗1-2遍,再用300μL PBS重悬。使用流式细胞仪检测荧光强度。
实施例3
没食子酸乙酯在治疗宫颈炎的实验研究:
目的:研究没食子酸乙酯对宫颈炎大鼠的治疗作用;
方法:采用25%苯酚胶浆构建结肠炎模型,造模成功后随机分空白组、模型组、阳性药组和没食子酸乙酯给药组;造模12天后,对大鼠外阴红肿和阴道分泌物进行评分、检测宫颈重量及脏器指数、检测大鼠血清中的瘤坏死因子α(TNF-α)、表皮生长因子(EGF)和表皮生长因子受体(EGFR)的表达情况,并对宫颈组织进行病理学切片;
结果:模型组大鼠阴道口红肿,有粘性分泌物流出,毛色变差,食欲下降;与模型组相比,给予药物治疗后,阳性药组、没食子酸乙酯组的大鼠逐渐恢复了行动和饮食,且大鼠阴道红肿和阴道分泌物的情况逐渐好转,红肿逐渐消失,阴道分泌物逐渐减少甚至消失。没食子酸乙酯可以抑制宫颈脏器指数的增加。病理组织切片表明宫颈炎大鼠宫颈组织上皮厚度增加,角化层脱落,粘膜及其固有层充血水肿,较多炎细胞浸润,腔内有大量炎性渗出物及坏死组织;与模型组相比,阳性药组、没食子酸乙酯组的宫颈组织病变减轻,病变处的部分组织已经开始得到修复,炎性细胞的浸润减轻,炎性渗出减少。经给药治疗后,较模型组来说,各治疗组大鼠的肿瘤坏死因子-α(TNF-α)水平显著降低(P<0.001)。没食子酸乙酯治疗显著增加表皮生长因子(EGF)和表皮生长因子受体(EGFR)的表达(P<0.001);结论:没食子酸乙酯对宫颈炎大鼠有明显的治疗作用;
25%苯酚胶浆和消糜栓胶浆的配制:
取苯酚2.5g、阿拉伯树胶粉7.5g,加蒸馏水至10mL,配成25%的苯酚胶浆;取3g消糜栓加热溶解,加阿拉伯树胶粉45g,加蒸馏水至9mL,配成0.33g/mL的阳性药;
动物分组及造模:
SD大鼠(洁净级,180±20g,雌性,未孕),由新疆维吾尔自治区疾病预防控制中心提供(SCXK(新)2014-0001);食用标准颗粒状饲料,适应性饲养1周后进行实验,随机分为空白组(a)、模型组(b)、阳性药组(c)、没食子酸乙酯给药组(350mg/kg)(d),每组6只,采用25%的苯酚胶浆诱导大鼠的宫颈炎模型,除空白组外,其他大鼠均阴道给药,用1mL注射器(针头换为灌胃针)插入阴道约2cm处,给予25%的苯酚胶浆0.15mL/只,3天1次,共4次,12天;空白组的大鼠则给予等量生理盐水替代25%苯酚胶浆;
给药:
造模结束后,不同组接受不同的治疗:阳性对照组每天给予270mg/kg的消糜栓胶浆;没食子酸乙酯给药组每天给予350mg/kg的消糜栓胶浆;空白组和模型组采用相似的阴道给药途径给予等体积生理盐水;所有大鼠采取阴道给药的方式连续给药7天;最后一次给药后处死动物,切除并收集宫颈组织;
一般情况观察:
从造模当天开始,观察并记录大鼠宫颈口和阴道分泌物,查看大鼠是否出现与空白组大鼠不一样的现象,如扎堆、少动、竖毛等现象,评分标准见表1;
表1外阴红肿和阴道分泌物评分标准
宫颈重量及脏器指数:
最后一次给药后次日,将动物脱颈椎处死,取出宫颈组织,称量宫颈子宫阴道部开口至双子宫分叉处的重量,并记录;
组织病理学检查:
从每组随机取出4个宫颈组织样本,置于的浓度10%的中性福尔马林溶液中进行固定,常规脱水,石蜡包埋,纵向切片,5μm厚,进行苏木精-伊红(HE)染色,在显微镜下进行观察并分析,综合粘膜变性坏死、脱落、溃疡、脓肿,炎细胞浸润等情况进行评分,具体评分参考见表2;
表2组织病理学评分标准
大鼠宫颈组织肿瘤坏死因子-α(TNF-α)、表皮生长因子(EGF)和表皮生长因子受体(EGFR)
指标检测:
处死大鼠前取血,分离血清3000rmp/min离心10min,-20℃保存备用,检测大鼠血清中的肿瘤坏死因子-α(TNF-α)、表皮生长因子(EGF)和表皮生长因子受体(EGFR)的表达情况,根据试剂盒说明书进行操作;
实验结果:
图1是五倍子水提液图及液质联用纯化系统分离得到的五倍子各成分,结合文献分析知,质荷比169.07为没食子酸、331.20为一-O-没食子酰基葡萄糖、483.26为二-O-没食子酰基葡萄糖、321.09为双没食子酸乙酯、635.34为三-O-没食子酰基葡萄糖、197.07为没食子酸乙酯、787.24为四-O-没食子酰基葡萄糖、939.27为五-O-没食子酰基葡萄糖、1090.63为六-O-没食子酰基葡萄糖、349.10为双没食子酸乙酯、1243.30为七-O-没食子酰基葡萄糖;
图2是五倍子各成分对细胞生长活力影响,表明一-O-没食子酰基葡萄糖、二-O-没食子酰基葡萄糖、双没食子酸、三-O-没食子酰基葡萄糖、没食子酸乙酯、四-O-没食子酰基葡萄糖的安全浓度高25μg/mL;双没食子酸乙酯和五-O-没食子酰基葡萄糖的安全浓度为6.25μg/mL;没食子酸和七-O-没食子酰基葡萄糖的安全浓度为3.125μg/mL;然而在这个浓度范围内六-O-没食子酰基葡萄糖具有细胞毒性;
图3是一-O-没食子酰基葡萄糖、二-O-没食子酰基葡萄糖、双没食子酸、三-O-没食子酰基葡萄糖、没食子酸乙酯、四-O-没食子酰基葡萄糖对细胞内一氧化氮(NO)表达的影响,研究表明一-O-没食子酰基葡萄糖、二-O-没食子酰基葡萄糖、双没食子酸、三-O-没食子酰基葡萄糖、没食子酸乙酯可以明显降低一氧化氮(NO)的表达(P<0.01);
图4是一-O-没食子酰基葡萄糖、二-O-没食子酰基葡萄糖、双没食子酸、三-O-没食子酰基葡萄糖、没食子酸乙酯、四-O-没食子酰基葡萄糖对细胞内白介素-6(IL-6)表达的影响,研究表明一-O-没食子酰基葡萄糖、二-O-没食子酰基葡萄糖、三-O-没食子酰基葡萄糖和没食子酸乙酯可以明显降低白介素-6(IL-6)的表达(P<0.01);
图5是一-O-没食子酰基葡萄糖、二-O-没食子酰基葡萄糖、双没食子酸、三-O-没食子酰基葡萄糖、没食子酸乙酯、四-O-没食子酰基葡萄糖对细胞内白介素-1β(IL-1β)表达的影响,研究表明二-O-没食子酰基葡萄糖、双没食子酸、三-O-没食子酰基葡萄糖和没食子酸乙酯可以明显降低白介素-1β(IL-1β)的表达(P<0.01);
图6是一-O-没食子酰基葡萄糖、二-O-没食子酰基葡萄糖、双没食子酸、三-O-没食子酰基葡萄糖、没食子酸乙酯、四-O-没食子酰基葡萄糖对细胞内肿瘤坏死因子-α(TNF-α)表达的影响,研究表明二-O-没食子酰基葡萄糖、三-O-没食子酰基葡萄糖和没食子酸乙酯可以明显降低肿瘤坏死因子-α(TNF-α)的表达(P<0.01);
图7是二-O-没食子酰基葡萄糖、三-O-没食子酰基葡萄糖和没食子酸乙酯对细胞内活性氧(ROS)产生的影响,没食子酸乙酯在6.25μg/mL、12.5μg/mL和25μg/mL可以明显降低细胞内活性氧(ROS)产生(P<0.01);二-O-没食子酰基葡萄糖和三-O-没食子酰基葡萄糖在25μg/mL降低细胞内活性氧(ROS)产生(P<0.05);
图8是没食子酸乙酯治疗宫颈炎大鼠的一般情况和脏器指数,模型组大鼠阴道口红肿,有粘性分泌物流出,毛色变差,食欲下降;与模型组相比,给予药物治疗后,阳性药组、没食子酸乙酯组的大鼠逐渐恢复了行动和饮食,且大鼠阴道红肿和阴道分泌物的情况逐渐好转,红肿逐渐消失,阴道分泌物逐渐减少甚至消失。没食子酸乙酯可以抑制宫颈脏器指数的增加;
图9是宫颈的组织病理学切片及组织病理学评分,宫颈炎大鼠宫颈组织上皮厚度增加,角化层脱落,粘膜及其固有层充血水肿,较多炎细胞浸润,腔内有大量炎性渗出物及坏死组织;与模型组相比,阳性药组、没食子酸乙酯组的宫颈组织病变减轻,病变处的部分组织已经开始得到修复,炎性细胞的浸润减轻,炎性渗出减少;
图10是没食子酸乙酯对大鼠宫颈组织肿瘤坏死因子-α(TNF-α)、表皮生长因子(EGF)和表皮生长因子受体(EGFR)的影响,经给药治疗后,较模型组来说,各治疗组大鼠的肿瘤坏死因子-α(TNF-α)水平显著降低(P<0.001)。没食子酸乙酯治疗显著增加表皮生长因子(EGF)和表皮生长因子受体(EGFR)的表达(P<0.001)。
Claims (2)
1.一种五倍子中抗炎活性成分没食子酸乙酯的筛选方法,其特征在于按下列步骤进行:
a、选用漆树科植物盐肤木、青麸杨或红麸杨叶上的虫瘿作为五倍子药材;
b、将步骤a中五倍子药材粉碎过60目筛,称取100 g药材粉末,以300 mL蒸馏水为溶剂回流提取3次,每次提取2小时,合并提取液;
c、将步骤b得到的合并提取液,使用X Bridge C18色谱柱对五倍子中的各成分进行纯化,分离得到的五倍子中11种成分包含:没食子酸,一-O-没食子酰基葡萄糖,二-O-没食子酰基葡萄糖,双没食子酸,三-O-没食子酰基葡萄糖,没食子酸乙酯,四-O-没食子酰基葡萄糖,五-O-没食子酰基葡萄糖,六-O-没食子酰基葡萄糖,双没食子酸乙酯和七-O-没食子酰基葡萄糖;
d、通过体外实验筛选五倍子中抗炎活性成分:以脂多糖诱导RAW264.7巨噬细胞形成炎症模型,通过噻唑蓝法测定五倍子中各成分对RAW264.7巨噬细胞生长活力的影响以及通过酶联免疫吸附法检测RAW264.7细胞上清液中一氧化氮、白介素-6、白介素-1β和肿瘤坏死因子-α的表达以及流式细胞术检测RAW264.7细胞中活性氧表达的影响,实验筛选出五倍子中抗炎活性成分为没食子酸乙酯。
2.根据权利要求1所述方法获得的五倍子中抗炎活性成分没食子酸乙酯在制备治疗宫颈炎的药物中的用途。
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