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医药导报
医药导报, 2016, 35(7): 705-709
doi: 10.3870/j.issn.1004-0781.2016.07.006
赖氨甘草次酸增溶及对结肠癌HCT-8细胞增殖抑制作用
Solubilization and Inhibitory Action of Glycyrrhetinic Acid Lysinate on Cell Proliferation of Coloretal Cancer Cell Line HCT-8
张丽娟1,, 钟家亮2, 古同男1, 魏秋芬1,
1.首都医科大学燕京医学院基础医学系,北京 101300
2.上海医药工业研究院,上海 200040
ZHANG Lijuan1,, ZHONG Jialiang2, GU Tongnan1, WEI Qiufen1,
1.Department of Basic Medicine, Yanjing Medical College,Capital Medical University,Beijing 101300,China
2.Shanghai Institute of Pharmaceutical Industry,Shanghai 200040,China

作者简介: 张丽娟(1970-),女,湖北公安人,副教授,硕士,主要从事中药作用机制与分子生物学研究。电话:010-69427565,E-mail:lijuanzh@sohu.com。

通信作者: 魏秋芬(1963-),女,北京顺义人,副教授,硕士,主要从事医学原虫的分子生物学与免疫学研究。电话:010-81487384,E-mail:weiqiuf@tom.com。
基金资助: 北京市教育委员会科技发展计划面上项目(KM200910025014)
中图分类号: R285     文献标识码: A     文章编号: 1004-0781(2016)07-0705-05
摘要:

目的 制备赖氨甘草次酸,研究其增溶作用及其对结肠癌HCT-8细胞增殖的抑制作用。方法 采用共研磨法制备赖氨甘草次酸,采用粉末X射线衍射法与红外光谱法对制备的赖氨甘草次酸进行表征,采用高效液相色谱法测定赖氨甘草次酸的增溶作用,采用噻唑蓝(MTT)法评价赖氨甘草次酸对结肠癌HCT-8细胞增殖的抑制作用。结果 制备获得赖氨甘草次酸。通过与赖氨酸成盐,甘草次酸溶解度提高约260倍,对结肠癌HCT-8细胞增殖的抑制作用提高约7倍。结论 赖氨甘草次酸较甘草次酸溶解度高,抗结肠癌HCT-8细胞效果好。
关键词: 赖氨甘草次酸 ; 增溶 ; 色谱法 ; 高效液相 ; 抗肿瘤活性

Abstract:

Objective To prepare,characterize glycyrrhetinic acid lysinate,and study the solubilization and inhibitory action antitumor activity of glycyrrhetinic acid lysinate on cell proliferation of colorectal cancer cell line HCT-8. Methods Glycyrrhetinic acid lysinate was prepared by co-grinding glycyrrhetinic acid with lysine in 1:1 molar mixture for 10 hours.Characterization of glycyrrhetinic acid lysinate was achieved by X-ray powder diffraction,infrared spectroscopy,and ultraviolet spectrum techniques.HPLC method was used to study the solubilization of glycyrrhetinic acid lysinate.The MTT method was used to assay the inhibitory action of glycyrrhetinic acid lysinate on cell proliferation. Results The solubility of glycyrrhetinic acid lysinate was enhanced 260 folds,compared with glycyrrhetinicacid in water.The inhibitory cell proliferation action on HCT-8 of glycyrrhetinic acid lysinate was 7 times higher than that of glycyrrhetinic acid. Conclusion The satisfactory water solubility and antitumor activity of glycyrrhetinic acid lysinate will be potentially useful for its application as a new pharmaceutical formulation in cancer treatment in the future.
Key words: Glycyrrhetinic acid lysinate ; Solubilization ; Chromatography ; high performance liquid ; Antitumor activity

从天然产物中寻找新的生物活性先导化合物是当前抗肿瘤药物研发的热点,1981—2002年,美国食品药品管理局(FDA)批准的抗肿瘤药有60%来源于天然产物[1]。甘草次酸是来源于甘草(Glycyrrhiza glabra)的具有多种生物活性的五环三萜类化合物。研究发现,甘草次酸可通过多种机制对肝细胞癌[2]、胃癌[3]、胰腺癌[4]、卵巢癌[5]、白血病[6]等多种肿瘤细胞产生不同程度的拮抗作用。甘草次酸及其衍生物还具有抗炎[7]、抗菌和抗病毒[8]、抗氧化[9]、保肝抗纤维化[10]、保护中枢神经[11]、保护肾脏[12]等多方面活性。但甘草次酸难溶于水,限制了其临床应用。

成盐修饰作为化学结构修饰的一项重要内容,因其诸多优点而引起广泛的研究与应用,具有开发新药的巨大潜在价值。自GOTTFRIED 等[13]在20世纪60年代合成甘珀酸钠用于治疗胃溃疡以来,国内外科学家从其母体结构入手进行化学修饰和结构改造,设计合成了许多有效的甘草次酸衍生物[14-17]。笔者利用赖氨酸与甘草次酸成盐的特性,制备赖氨甘草次酸并考察其对人结肠癌HCT-8肿瘤细胞的体外抑制作用。

1 材料与方法
1.1 药品与试剂

甘草次酸[南京泽朗医药科技有限公司,高效液相色谱(HPLC)法测得纯度≥98.5%,批号:20100807],赖氨酸(北京欣经科生物技术有限公司,批号:101205),甘草次酸对照品(中国食品药品检定研究院,批号:110723-201413),甲醇(J&K CHEMICAL LTD,HPLC级,批号:20141017),娃哈哈饮用纯净水(杭州娃哈哈集团有限公司)。其余试剂均为分析纯。人结肠癌细胞株HCT-8购自中国医学科学院肿瘤医院,RPMI-1640培养液购自上海博耀生物科技有限公司。

1.2 仪器与设备

Bruker D8 ADVANCE 粉末衍射仪(德国布鲁克);Nicolet 5700型傅里叶变换红外光谱仪(美国尼高力);TU1901双光束紫外-可见分光光度仪(北京普析通用仪器有限公司);Angilent 1200高效液相色谱仪(美国安捷伦),WD-2102A型酶标仪(北京六一仪器厂)。

1.3 赖氨甘草次酸的制备

取甘草次酸2 353.40 mg,赖氨酸730.95 mg,混合均匀,置于球磨机中研磨10 h,即得赖氨甘草次酸。

1.4 赖氨甘草次酸的表征

1.4.1 粉末X射线衍射实验 X射线衍射图谱的摄取:采用粉末衍射仪测定,测定条件为 vertical goniometer,Ni-filter单色器,Theta/Theta连续扫描,电压40 kV,电流40 mA,扫描速度8 °·min-1,LynxEye 检测器,扫描范围3 °~80 °。

1.4.2 傅里叶红外光谱分析实验 样品采用衰减全反射傅里叶变换红外光谱仪(ATR-FT-IR)测定,光谱范围4 000~650 cm-1,分辨率4.000 cm-1,扫描16次,测定红外吸收光谱。

1.4.3 紫外光谱分析 样品采用紫外分光光度仪测定,波长扫描范围:200~400 nm;扫描方式:单次扫描;扫描速度:中速;扫描间隔:1.0 nm。

1.4.4 HPLC分析 色谱柱为Agilent Eclipse XDB C18(250 mm×4.6 mm,5 μm);流动相为甲醇:0.5%醋酸=85:15;流速:1.0 mL·min-1;柱温30 ℃;进样量:10 μL;检测波长249 nm。

1.4.5 体外噻唑蓝(MTT)比色法测定对结肠癌HCT-8细胞增殖的抑制活性 采用人结肠癌细胞株HCT-8,以含10%胎牛血清,100 U·mL-1青霉素、100 μg·mL-1链霉素的RPMI-1640培养液,在37 ℃、5% CO2下培养,24 h后细胞贴壁生长,每3~4 d传代一次,取对数生长期的HCT-8细胞制成2×105 个·mL-1细胞悬液,加入96孔培养板中,每孔200 μL。待24 h细胞贴壁生长后弃去培养液。

活性测定:各化合物先用二甲亚砜(DMSO)溶解,再经RPMI-1640培养液稀释至终浓度为5,10,20,50,100,300 μg·mL-1,空白孔加入RPMI-1640培养液,对照组加入0.1%DMSO。阳性药物采用氟尿嘧啶(5-FU)。实验中每个浓度设3个平行孔,药物干预48 h后用MTT法[18-19]经酶标仪在490 nm波长处测定吸光度(A)值,空白孔调零,取平均值。按照下列公式计算细胞生长抑制率[20]:

生长抑制率(%)=(1- 实验组平均 A 值 对照组平均 A 值 )×100%。

2 结果
2.1 粉末X射线衍射分析

粉末X射线衍射图谱见图1。


图1 粉末X射线衍射图谱

Fig.1 Spectra of powder X-ray diffraction analysis

由图1可见,甘草次酸是晶态化合物,因而有尖锐的特征衍射峰;赖氨酸属于结晶度不好的晶态化合物,赖氨酸、甘草次酸(1:1)物理混合物的主要衍射峰包含甘草次酸和赖氨酸的衍射峰,为两种成分衍射峰的物理叠加;赖氨甘草次酸为非晶态化合物,因而无明显尖锐衍射峰,表现为弥散衍射峰,表明通过制备过程赖氨酸与甘草次酸形成了新的物相赖氨甘草次酸。

2.2 紫外光谱分析

原料赖氨酸的最大吸收峰在波长204 nm,甘草次酸则在249 nm 有一最大吸收峰,系分子结构中α,β-不饱和羰基共轭双键的特征吸收峰。赖氨甘草次酸的紫外最大吸收峰为249 nm,与甘草次酸无差异,吸收强度略降低,表明成盐作用对甘草次酸的共轭吸收系统影响不大。

2.3 傅里叶红外光谱分析

样品的ATR-FT-IR测定结果见图2。


图2 ATR-FT-IR测定图谱

Fig.2 ATR-FT-IR spectra

甘草次酸具有齐墩果烷型骨架,分子中存在特征的偕二甲基、α,β-不饱和酮及羧基,这些结构在其IR谱图上均可得到确认。1 384 cm-1和1 366 cm-1处的特征双峰为C-4连接的偕二甲基的C-H面内弯曲振动吸收;1 701 cm-1附近为C-30羧基的υC=O吸收;由于受双键共轭的影响,C-11羰基的υC=O略向低波数方向位移至1 659 cm-1附近。此外,在3 434 cm-1和986 cm-1附近分别可见C-3羟基的伸缩振动吸收和面外弯曲振动吸收。赖氨酸在IR 谱图上可见-NH3的弯曲振动吸收(1 573 cm-1)和-COO-的对称伸缩振动吸收(1 404 cm-1),和原料相比,赖氨酸与甘草次酸物理混合物的红外光谱差别不大。赖氨甘草次酸的IR谱图中,C-30羧基υC=O吸收减弱,与C-11羰基υC=O重叠,使此处的吸收有所增强。IR分析结果提示,成盐反应是通过甘草次酸C-30羧基与赖氨酸结构中的氨基进行。

2.4 增溶作用分析

2.4.1 标准曲线的建立 取甘草次酸对照品25.06 mg精密称定,移至25 mL量瓶中,甲醇定容,配制成1 002.4 μg·mL-1储备液。精密吸取0.01,0.1,0.5,1.0,2.5,5.0 mL至10 mL量瓶,用流动相稀释至刻度,获得6种浓度样品,分别为1.00,10.02,50.12,100.24,250.60,501.00 μg·mL-1。分别精密吸取6种浓度溶液与储备液各10 μL注入液相色谱仪,重复进样3次,记录色谱图及峰面积,以样品浓度数据为横坐标,以检测获得的峰面积为纵坐标,绘制标准曲线。甘草次酸溶液在1.00~1 002.40 μg·mL-1浓度范围内,回归线性方程为:Y = 14.057X+21.984,相关系数R2=1.000 0(n=7),表明利用HPLC法建立的甘草次酸吸收峰面积与浓度间呈现良好的线性关系,可作为甘草次酸定值检测分析方法。

2.4.2 精密度实验 精密量取501.00 μg·mL-1甘草次酸溶液10 μL注入液相色谱仪,重复进样6次,记录色谱图及峰面积,结果6份样品平均峰面积RSD值为0.10%,表明利用HPLC法测定甘草次酸含量的的检测分析仪器精密度良好。

2.4.3 重复性实验 取6份甘草次酸对照品各12.50 mg精密称定,移至25 mL量瓶中,使用甲醇定容,摇匀,备用。分别精密吸取供试品溶液10 μL注入液相色谱仪,每个样品平行进样3次。记录色谱图及峰面积,计算得6份样品平均峰面积RSD为0.25%,表明利用HPLC法测定甘草次酸含量的检测分析方法具有良好的精密度。

2.4.4 稳定性实验 精密量取501.00 μg·mL-1甘草次酸溶液,分别于0,1,2,4,8,12 h取10 μL注入液相色谱仪,每个样品平行进样3次。记录色谱图及峰面积,计算获得稳定性。测定RSD值为1.12%,表明利用HPLC法测定甘草次酸含量的检测分析方法在12 h内含量测定的稳定性良好。

2.4.5 方法回收率实验 按照《中华人民共和国药典》2010年版规定,取甘草次酸原料药适量精密称定,按80%,100%,120%比例加入对照品适量,配制成相应浓度溶液,取10 mL注入液相色谱仪,记录色谱图及峰面积,计算平均回收率,结果见表1。方法平均回收率98.94%,RSD为0.99%,处于98%~102%范围内,表明高效液相色谱法测定甘草次酸含量的检测分析方法准确度良好。

表1 甘草次酸回收率实验结果
Tab.1 Result of recovery experiment of glucyrrhetinic acid mg
原有量 加入量 测得量 回收率/%
12.43 9.97 22.49 100.90
12.41 9.96 22.32 99.49
12.41 9.96 22.20 98.29
12.41 12.41 24.75 99.44
12.42 12.41 24.65 98.55
12.41 12.41 24.65 98.63
12.41 14.97 27.10 98.13
12.43 14.98 27.29 99.20
12.42 14.98 27.05 97.79

表1 甘草次酸回收率实验结果

Tab.1 Result of recovery experiment of glucyrrhetinic acid mg

2.4.6 溶解度实验 取适量甘草次酸、赖氨甘草次酸、赖氨酸-甘草次酸物理混合物,分别制备饱和溶液,滤纸过滤,孔径0.22 μm微孔滤膜滤过备用。分别将甘草次酸、赖氨甘草次酸、赖氨酸-甘草次酸物理混合物的饱和溶液取样10 μL进样,重复测定3次,取平均值,以峰面积计算甘草次酸含量,结果见表2。

表2 甘草次酸含量测定结果
Tab.2 Content determination of glycyrrhetinic acid
序号 样品 峰面积 浓度/
(μg·mL-1)
1 甘草次酸饱和溶液 36.8 1.05
2 赖氨酸-甘草次酸物理混合物饱和溶液 282.8 18.55
3 赖氨甘草次酸饱和溶液 4 000.7 283.06

表2 甘草次酸含量测定结果

Tab.2 Content determination of glycyrrhetinic acid

测定结果表明,甘草次酸几乎不溶于水。赖氨酸可以增加甘草次酸的吸水性,因此其物理混合物的溶解度略有增加。通过赖氨酸的水溶性,制备获得的赖氨甘草次酸的溶解度为283.06 μg·mL-1,其溶解度提高了约260倍,明显改善了甘草次酸的水溶性。

2.5 对结肠癌HCT-8细胞增殖的抑制作用

药物浓度在5~300 μg·mL-1范围内,赖氨甘草次酸对肿瘤细胞显示明显的抑制作用,对结肠癌HCT-8肿瘤细胞增殖的抑制率为10.7%~87.8%,细胞增殖抑制率随药物浓度的增加而逐渐上升。在药物浓度为20与50 μg·mL-1条件下,赖氨甘草次酸对肿瘤细胞的抑制活性与阳性对照药5-FU相比,差异无统计学意义,与甘草次酸相比,差异有统计学意义(表3)。

表3 赖氨甘草次酸对HCT-8细胞抑制作用测定结果
Tab.3 Detection results of the inhibition of glycyrrhetinic acid lysinate on HCT-8 cells x¯±s,n=3
药物与浓度/
(μg·mL-1)		 抑制率/
%		 药物与浓度/
(μg·mL-1)		 抑制率/
%
5-FU 50 30.0±2.9
5 23.6±2.8 100 45.1±2.8
10 45.2±2.7 300 60.9±2.6
20 65.4±2.7 赖氨甘草次酸
50 80.5±2.1 5 10.7±0.9
100 100.0±0.0 10 30.7±2.0
300 100.0±0.0 20 62.5±2.6*1
甘草次酸 50 78.2±2.4*1
5 3.7±0.6 100 80.2±1.9
10 5.5±0.8 300 87.8±2.6
20 10.4±1.1

Compared with glycyrrhetinic acid at the same concentration,*1P<0.05

与同浓度甘草次酸比较,*1P<0.05

表3 赖氨甘草次酸对HCT-8细胞抑制作用测定结果

Tab.3 Detection results of the inhibition of glycyrrhetinic acid lysinate on HCT-8 cells x¯±s,n=3

3 讨论

甘草次酸具有多种生物学活性,但其对结肠癌细胞株HCT-8的杀灭作用笔者尚未见报道,笔者在本实验中考察了甘草次酸与赖氨甘草次酸对HCT-8细胞的杀灭作用。结果表明,通过与赖氨酸成盐,甘草次酸对HCT-8细胞的杀灭作用提高约7倍,为将甘草次酸进一步开发为新高效低毒抗癌新药提供了思路。

The authors have declared that no competing interests exist.
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[2] SATOMI Y,NISHINO H,SHIBATA S.Glycyrrhetinic acid and related compounds induce G1 arrest and apoptosis in human hepatocellular carcinoma HepG2[J].Anticancer Res,2005,25(6B):4043-4047.
Glycyrrhetinic acid (GA) and its related compounds are known to have anti-inflammatory activity and also to inhibit liver carcinogenesis and tumor growth. GA and related compounds inhibited cell proliferation of the human hepatoma cell line, HepG2. Among five compounds tested, ursolic acid and 18beta-olean-12-ene-3beta, 23, 28-triol (18beta-erythrotriol) were comparatively effective, where the 50% inhibitory dose was 20 microM and 25 microM, respectively. Flow-cytometric analysis showed that GA and the related compounds arrested the cell cycle in the G1-phase; in addition, GA-related compounds induced apoptosis at high dose. Western blot analysis indicated that the induction of apoptosis by GA and ursolic acid was accompanied with an activation of caspase-8 and a reduction in the anti-apoptotic proteins, Bcl-2 and Bcl-xL, although the pro-apoptotic proteins, Bax and Bak, remained unaffected. These results suggest that GA and its related compounds may be potent agents in liver cancer treatment.
DOI:10.1039/b719498f      PMID:16309197      URL    
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[3] HIBASAMI H,IWASE H,YOSHIOKA K,et al.Glycyrrhetic acid(a metabolic substance and aglycon of glycyrrhizin) induces apoptosis in human hepatoma,promyelotic leukemia and stomach cancer cells[J].Int J Mol Med,2006,17(2):215-219.
We have investigated the effect of glycyrrhetic acid (GR) which is metabolic substance of glycyrrhizin, on DNA of human hepatoma (HLE), promyelotic leukemia (HL-60) and stomach cancer (KATO III) cells. GR displayed apoptotic effects against HLE, HL-60 and KATO III cells. The fragmentation of DNA by GR to oligonucleosomal-sized fragments, a characteristic of apoptosis, was dose- and time-dependent in these cell lines. These findings suggest that growth inhibition of these cell lines by GR result from the induction of apoptosis by the compound. Inhibitors of caspases did not suppress the DNA fragmentation caused by GR. N-acetyl-L-cysteine, an antioxidant drug, weakly inhibited the DNA fragmentation caused by GR suggesting that active oxidants work partly as an apoptosis-inducing transfer substance.
DOI:10.3892/ijmm.17.2.215      PMID:16391818      URL    
[本文引用:1]
[4] JUTOORU I,CHADALAPAKA G,CHINTHARLAPALLI S,et al.Induction of apoptosis and nonsteroidal anti-inflammatory drug-activated gene 1 in pancreatic cancer cells by a glycyrrhetinic acid derivative[J].Mol Carcinog,2009,48(8):692-702.
Methyl 2-cyano-3,11-dioxo-18beta-olean-1,12-dien-30-oate (CDODA-Me) is a synthetic triterpenoid derived from glycyrrhetinic acid, a bioactive phytochemical in licorice, CDODA-Me inhibits growth of Panc1 and Panc28 pancreatic cancer cell lines and activates peroxisome proliferator-activated receptor gamma (PPARgamma)-dependent transactivation in these cells. CDODA-Me also induced p21 and p27 protein expression and downregulates cyclin D1; however, these responses were receptor-independent. CDODA-Me induced apoptosis in Panc1 and Panc28 cells, and this was accompanied by receptor-independent induction of the proapoptotic proteins early growth response-1 (Egr-1), nonsteroidal anti-inflammatory drug-activated gene-1 (NAG-1), and activating transcription factor-3 (ATF3). Induction of NAG-1 and Egr-1 by CDODA-Me was dependent on activation of phosphatidylinositol-3-kinase (PI3-K) and/or p42 and p38 mitogen-activated protein kinase (MAPK) pathways but there were differences between Panc28 and Panc1 cells. Induction of NAG-1 in Panc28 cells was p38-MAPK- and PI3-K-dependent but Egr-1-independent, whereas induction in Panc1 cells was associated with activation of p38-MAPK, PI3-K, and p42-MAPK and was only partially Egr-1-dependent. This is the first report of the induction of the proapoptotic protein NAG-1 in pancreatic cancer cells.
DOI:10.1002/mc.20518      PMID:19125423      URL    
[本文引用:1]
[5] LEE C S,YANG J C,KIM Y J,et al.18β-Glycyrrhetinic acid potentiates apoptotic effect of trichostatin A on human epithelial ovarian carcinoma cell lines[J].Eur J Pharmacol,2010,649(1-3):354-361.
The licorice-derived compounds glycyrrhizin and 18β-glycyrrhetinic acid have been shown to induce apoptosis in various cancer cells. However, the effect of these licorice compounds on the apoptotic effect of histone deacetylase inhibitors in epithelial ovarian carcinoma cells has not been determined. We assessed the effect of 18β-glycyrrhetinic acid on trichostatin A-induced apoptosis in the human epithelial carcinoma cell lines OVCAR-3 and SK-OV-3. Trichostatin A induced nuclear damage, decreased Bid and Bcl-2 protein levels, increased in Bax levels, induced cytochrome c release, activated caspase-8, -9 and -3, and increased tumor suppressor p53 levels. 18β-Glycyrrhetinic acid potentiated the trichostatin A-induced apoptosis-related protein activation and cell death. Unlike 18β-glycyrrhetinic acid, up to 25 μM of the pro-compound glycyrrhizin did not induce cell death and did not affect trichostatin A-induced apoptosis. The results suggest that 18β-glycyrrhetinic acid may potentiate the apoptotic effects of trichostatin A against ovarian carcinoma cell lines by increasing the activation of the caspase-8-dependent pathway as well as the activation of the mitochondria-mediated cell death pathway, leading to activation of caspases. 18β-Glycyrrhetinic acid may enhance the therapeutic effect of trichostatin A against epithelial ovarian adenocarcinoma. Copyright 08 2010 Elsevier B.V. All rights reserved.
DOI:10.1016/j.ejphar.2010.09.047      PMID:20868669      URL    
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[6] HIBASAMI H,IWASE H,YOSHIOKA K,et al.Glycyrrhizin induces apoptosis in human stomach cancer KATO III and human promyelotic leukemia HL-60 cells[J].Int J Mol Med,2005,16(2):233-236.
We have investigated the effects of (GL) on of stomach KATO III and promyelotic HL-60 , and on DNA of those lines. GL displayed inhibitory effect against KATO III and HL-60 . Morphological change showing was observed in the KATO III and HL-60 treated with GL. The fragmentation of DNA by GL to oligonucleosomal-sized fragments that is a characteristic of apoptosis was observed to be concentration- and time-dependent in both lines. inhibitors such as Z-VAD-FMK and Z-DCB suppressed the DNA fragmentation induced by GL. The data of the present study show that the suppression of KATO III and HL-60 by GL results from the induction of apoptosis by GL, and that is involved in the induction of apoptosis by GL in these .
DOI:10.3892/ijmm.16.2.233      PMID:16012754      URL    
[本文引用:1]
[7] MATSUI S,MATSUMOTO H,SONODA Y,et al.Glycyrr-hizin and related compounds down-regulate production of inflammatory chemokines IL-8 and eotaxin 1 in a human lung fibroblast cell line[J].Int Immunopharmacol,2004,4(13):1633-1644.
<h2 class="secHeading" id="section_abstract">Abstract</h2><p id="">Glycyrrhizin (GL) is known to have various immunomodulating activities and has long been used clinically as an anti-allergic and anti-hepatitis agent. While the potency of GL against lung inflammatory diseases has been expected, the effect of GL on the lung has been poorly understood. Lung fibroblasts are known as a potent producer of inflammatory chemokines, IL-8 and eotaxin 1, by which neutrophils and eosinophils are strongly attracted during inflammation. Therefore, we studied the effects of GL on the production of these chemokines using a human fetal lung fibroblast cell line, HFL-1, stimulated with TNF-α and IL-4. Moreover, we examined the structure&ndash;activity relationships of GL to explore more beneficial compounds. 18α,β-GL inhibited IL-8 dose-dependently and inhibited eotaxin 1 slightly. 18α,β-Glycyrrhetic acid (GA) did not inhibit IL-8 but inhibited eotaxin 1. The effect of 18α,β-glycyrrhetic acid monoglucuronide (MGA) resembled that of 18α,β-GL but was weaker. Both 3β-[(2-<em>O</em>-β-<span class="smallcaps">d</span>-glucopyranuronosyl-β-<span class="smallcaps">d</span>-glucopyranuronosyl)oxy]-18β-11-deoxo-olean-12-en-30-oic acid (11-deoxo-GL) and 3β-[(2-<em>O</em>-β-<span class="smallcaps">d</span>-glucopyranuronosyl-β-<span class="smallcaps">d</span>-glucopyranuronosyl)oxy]-olean-11,13,(18)-dien-30-oic acid (hetero-GL) exhibited inhibitory activity with significant cytotoxicity. 3β-[(2-<em>O</em>-β-<span class="smallcaps">d</span>-Glucopyranuronosyl-β-<span class="smallcaps">d</span>-glucopyranuronosyl)oxy]-18β-olean-9,12-dien-30-oic acid (homo-GL) did not have cytotoxicity but its activity was mild like that of 18α,β-GL. 3β-[(2-<em>O</em>-β-<span class="smallcaps">d</span>-Glucopyranuronosyl-β-<span class="smallcaps">d</span>-glucopyranuronosyl)oxy]-olean-11,13(18)-dien-30-ol (hetero-30-OH-GL) and 3β-[(2-<em>O</em>-β-<span class="smallcaps">d</span>-glucopyranuronosyl-β-<span class="smallcaps">d</span>-glucopyranuronosyl)oxy]-18β-olean-9,12-dien-30-ol (homo-30-OH-GL) showed potent inhibitory effects, at concentrations lower than 18α,β-GL with no significant cytotoxicity. These results suggest that GL-related compounds are effective in reducing chemokine production and that GL-modified compounds including hetero-30-OH-GL and homo-30-OH-GL appear most beneficial in view of their inhibitory capacity with less cytotoxicity.</p>
DOI:10.1016/j.intimp.2004.07.023      Magsci    
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[8] LIN J C,CHERNG J M,HUNG M S,et al.Inhibitory effects of some derivatives of glycyrrhizic acid against Epstein-Barr virus infection:structure-activity relationships[J].Antiviral Res,2008,79(1):6-11.
Glycyrrhizic acid (18β-GL or GL) is a herbal drug with a broad spectrum of antiviral activities and pharmacological effects and multiple sites of action. Previously we showed that GL inhibits Epstein-Barr virus (EBV) infection in vitro by interfering with an early step of the EBV replication cycle (possibly attachment/penetration). Here we tested the effects of 15 GL derivatives against EBV infection by scoring the numbers of cell expressing viral antigens and quantifying EBV DNA copy numbers in superinfected Raji cells. The derivatives were made either by transformation of GL on carboxyl and hydroxyl groups or by conjugation of amino acid residues into the carbohydrate part. We identified seven compounds active against EBV and all showed dose-dependent inhibition as determined by both assays. Among these active compounds, the introduction of amino acid residues into the GL carbohydrate part enhanced the antiviral activity in three of the seven active compounds. However, when Glu(OH)-OMe was substituted by Glu(OMe)-OMe, its antiviral activity was completely abolished. Introduction of potassium or ammonium salt to GL reduced the antiviral activity with no significant effect on cytotoxicity. The α-isomer (18α-GL) of 18β-GL was as potent as the β-form, but its sodium salt lost antiviral activity. The metabolic product of GL, 18β-glycyrrhetinic acid (18β-GA or GA), was 7.5-fold more active against EBV than its parental compound GL but, concomitantly, exhibited increased cytotoxicity resulting in a decreased therapeutic index.
DOI:10.1016/j.antiviral.2008.01.160      PMID:18423902      URL    
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[9] MAITRAIE D,HUNG C F,TU H Y,et al.Synthesis,anti-inflammatory,and antioxidant activities of 18beta-glycyrrhetinic acid derivatives as chemical mediators and xanthine oxidase inhibitors[J].Bioorg Med Chem,2009,17(7):2785-2792.
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[10] JEONG H G,YOU H J,PARK S J,et al.Hepatoprotective effects of 18beta-glycyrrhetinic acid on carbon tetrachloride-induced liver injury:inhibition of cytochrome P450 2E1 expression[J].Pharmacol Res,2002,46(3):221-227.
The protective effects of 18-glycyrrhetinic acid (GA), the aglycone of glycyrrhizin (GL) derived from licorice, on carbon tetrachloride-induced hepatotoxicity and the possible mechanisms involved in this protection were investigated in mice. Pretreatment with GA prior to the administration of carbon tetrachloride significantly prevented an increase in serum alanine, aspartate aminotransferase activity and hepatic lipid peroxidation in a dose-dependent manner. In addition, pretreatment with GA also significantly prevented the depletion of glutathione (GSH) content in the livers of carbon tetrachloride-intoxicated mice. However, reduced hepatic GSH levels and glutathione--transferase activities were unaffected by treatment with GA alone. Carbon tetrachloride-induced hepatotoxicity was also prevented, as indicated by a liver histopathologic study. The effects of GA on the cytochrome P450 (P450) 2E1, the major isozyme involved in carbon tetrachloride bioactivation, were also investigated. Treatment of mice with GA resulted in a significant decrease of the P450 2E1-dependent hydroxylation of-nitrophenol and aniline in a dose-dependent manner. Consistent with these observations, the P450 2E1 expressions were also decreased, as determined by immunoblot analysis. GA also showed antioxidant effects upon FeClascorbate-induced lipid peroxidation in mice liver homogenate and upon superoxide radical scavenging activity. These results show that protective effects of GA against the carbon tetrachloride-induced hepatotoxicity may be due to its ability to block the bioactivation of carbon tetrachloride, primarily by inhibiting the expression and activity of P450 2E1, and its free radical scavenging effects.
DOI:10.1016/S1043-6618(02)00121-4      PMID:12220964      URL    
[本文引用:1]
[11] CHERNG J M,LIN H J,HUNG M S,et al.Inhibition of nuclear factor kappaB is associated with neuroprotective effects of glycyrrhizic acid on glutamate-induced excitotoxicity in primary neurons[J].Eur J Pharmacol,2006,547(1-3):10-21.
2006.12.16 ~ 2006.12.17
URL    
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[12] AL-DUJAILI E A,KENYON C J,NICOL M R,et al.Li-quorice and glycyrrhetinic acid increase DHEA and deoxycorticosterone levels in vivo and in vitro by inhibiting adrenal SULT2A1 activity[J].Mol Cell Endocrinol,2011,336(1-2):102-109.
The mineralocorticoid effects of liquorice are mediated by the inhibitory effects of one of its active components glycyrrhetinic acid on 11 beta-hydroxysteroid dehydrogenase type 2. However, liquorice is reputed to have many medicinal properties and also contains a number of other potentially biologically active compounds. Here we have investigated the wider effects of oral liquorice on steroidogenesis focussing particularly on possible inhibitory effects of glycyrrhetinic acid on adrenal sulfotransferase activity.<br/>Salivary steroids were profiled by ELISA in groups of normal male and female volunteers after consuming either liquorice-containing or non-liquorice-containing confectionary for one week. Cortisol and cortisone levels reflected expected inhibition of 11 beta-hydroxysteroid dehydrogenase type 2 by glycyrrhetinic acid. Salivary aldosterone was decreased but deoxycorticosterone, dehydroepiandrosterone and testosterone were increased. To assess whether glycyrrhetinic acid directly affected steroidogenesis, free and conjugated steroids were measured in incubates of adrenocortical H295 cells, firstly, in the presence or absence of forskolin and secondly, with radiolabeled deoxycorticosterone or dehydroepiandrosterone. Glycyrrhetinic acid inhibited cortisone and enhanced cortisol synthesis consistent with 11 beta-hydroxysteroid dehydrogenase type 2 inhibition. Basal and forskolin-stimulated syntheses of deoxycorticosterone and dehydroepiandrosterone conjugates were also inhibited in a dose-dependent manner; glycyrrhetinic acid inhibited the conjugation of deoxycorticosterone and dehydroepiandrosterone with IC50 values of 7 mu M. Inhibition of deoxycorticosterone and dehydroepiandrosterone conjugation was apparent within 4 h of starting glycyrrhetinic acid treatment and was not associated with changes in the expression of SULT2A1 mRNA. SULT2A1 encodes the enzyme sulfotransferase 2A1 which is responsible for the sulfonation of deoxycorticosterone and dehydroepiandrosterone as well as pregnenolone and 17-hydroxypregnenolone in human adrenal glands. We suggest that the glycyrrhetinic acid constituent of liquorice increases circulating and thereby, salivary levels of unconjugated deoxycorticosterone and dehydroepiandrosterone by inhibiting their conjugation at source within the adrenal cortex. This effect may contribute to the mineralocorticoid actions of glycyrrhetinic acid and gives substance to claims that liquorice also has androgenic properties. (C) 2010 Elsevier Ireland Ltd. All rights reserved.
DOI:10.1016/j.mce.2010.12.011      Magsci    
[本文引用:1]
[13] GOTTFRIED S,LILY B.Giycyrrheinic acid derivatives[P].GB:843133,1960-08-04.
[本文引用:1]
[14] KONDRATENKO R M,MUSTAFMA S R,BALTINA L A,et al.Synthesis and antiulcer activity of 3-O-acylated glycyrrhetic acid methylates[J].Pharm Chem J,2001,35(5):243-246.
Abstract: No Abstract
DOI:10.1023/A:1011905103576      URL    
[本文引用:1]
[15] SCHWARZ S,CSUK R.Synthesis and antitumour activity of glycyrrhetinic acid derivatives[J].Bioorg Med Chem,2010,18(21):7458-7474.
Glycyrrhetinic acid (GA) is one of many interesting triterpenoic acids showing anticancerogenic potential. GA is known to trigger apoptosis in tumour cell lines, although GA has a low cytotoxicity. In our study we were able to prepare derivatives of GA that show lowered the IC 50 values as determined by a sulforhodamine B (SRB) assay using 15 different human tumour cell lines. Thus, combining an ester group combined with the presence of an amino acid moiety led to a ca. 60-fold improved antitumor activity. Experiments on mouse embryonic fibroblasts (NiH3T3) revealed that these compounds showed a better selectivity for tumour cells compared to the parent compound GA. An apoptotic effect of some of these compounds was determined using an acridine orange/ethidium bromide (AO/EB) test and DNA laddering experiments.
DOI:10.1016/j.bmc.2010.08.054      PMID:20932766      URL    
[本文引用:0]
[16] PARIDA P K,SAU A,GHOSH T,et al.Synthesis and eva-luation of triazole linked glycosylated 18β-glycyrrhetinic acid derivatives as anticancer agents[J].Bioorg Med Chem Lett,2014,24(16):3865-3868.
A series of glycosyl triazol linked 18β-glycyrrhetinic acid (GA) derivatives have been synthesized using 1,3-dipolar cycloaddition reaction of per-O-acetylated glycosyl azide derivatives ( 4a – h ) with propargyl ester of 18β-glycyrrhetinic acid (GA) ( 2 and 3 ) following the concept of ‘Click chemistry’. The synthesized triazole derivatives were de-O-acetylated to furnish compounds ( 7a – h and 8a – c ) with free hydroxyl groups in the carbohydrate moieties, which were evaluated for their anticancer potential against human cervical cancer cells (HeLa) and normal kidney epithelial (NKE) cells. GA ( 1 ), compound 7d, compound 7g and compound 8c showed promising anticancer activities.
DOI:10.1016/j.bmcl.2014.06.054      PMID:25027936      URL    
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[17] SCHWARZ S,LUCAS S D,SOMMEERWERK S,et al.Am-ino derivatives of glycyrrhetinic acid as potential inhibitors of cholinesterases[J].Bioorg Med Chem,2014,22(13):3370-3378.
The development of remedies against the Alzheimer鈥檚 disease (AD) is one of the biggest challenges in medicinal chemistry nowadays. Although not completely understood, there are several strategies fighting this disease or at least bringing some relief. During the progress of AD, the level of acetylcholine (ACh) decreases; hence, a therapy using inhibitors should be of some benefit to the patients. Drugs presently used for the treatment of AD inhibit the two ACh controlling enzymes, acetylcholinesterase as well as butyrylcholinesterase; hence, the design of selective inhibitors is called for. Glycyrrhetinic acid seems to be an interesting starting point for the development of selective inhibitors. Although its glycon, glycyrrhetinic acid is known for being an AChE activator, several derivatives, altered in position C-3 and C-30, exhibited remarkable inhibition constants in micro-molar range. Furthermore, five representative compounds were subjected to three more enzyme assays (on carbonic anhydrase II, papain and the lipase from Candida antarctica ) to gain information about the selectivity of the compounds in comparison to other enzymes. In addition, photometric sulforhodamine B assays using murine embryonic fibroblasts (NiH 3T3) were performed to study the cytotoxicity of these compounds. Two derivatives, bearing either a 1,3-diaminopropyl or a 1 H -benzotriazolyl residue, showed a BChE selective inhibition in the single-digit micro-molar range without being cytotoxic up to 30渭M. In silico molecular docking studies on the active sites of AChE and BChE were performed to gain a molecular insight into the mode of action of these compounds and to explain the pronounced selectivity for BChE.
DOI:10.1016/j.bmc.2014.04.046      PMID:24853320      URL    
[本文引用:1]
[18] YOUNG F M,PHUNGTAMDET W,SANDERSON B J.Mo-dification of MTT assay conditions to examine the cytotoxic effects of amitraz on the human lymphoblastoid cell line,WIL2NS[J].Toxicol In Vitro,2005,19(8):1051-1059.
[本文引用:1]
[19] SEIDL K,ZINKERNAGEL A S.The MTT assay is a rapid and reliable quantitative method to assess Staphylococcus aureus induced endothelial cell damage[J].J Microbiol Methods,2013,92(3):307-309.
We established the 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay to assess damage induced by Staphylococcus aureus in human umbilical vein endothelial cells (HUVECs). The MTT assay was comparable to the previously published (51)chromium release assay. MTT reduction by intracellular S. aureus was negligible and therefore permits assessment of S. aureus induced EC damage.
DOI:10.1016/j.mimet.2012.12.018      PMID:23275136      URL    
[本文引用:1]
[20] LI L,SHANG B,HU L,et al.Site-specific PEGylation of lidamycin and its antitumor activity[J].Acta Pharm Sin B,2015,5(3):264-269.
In this study, N-terminal site-specific mono-PEGylation of the recombinant lidamycin apoprotein (rLDP) of lidamycin (LDM) was prepared using a polyethyleneglycol (PEG) derivative ( M w 20聽kDa) through a reactive terminal aldehyde group under weak acidic conditions (pH 5.5). The biochemical properties of mPEG-rLDP-AE, an enediyne-integrated conjugate, were analyzed by SDS-PAGE, RP-HPLC, SEC-HPLC and MALDI-TOF. Meanwhile, in vitro and in vivo antitumor activity of mPEG-rLDP-AE was evaluated by MTT assays and in xenograft model. The results indicated that mPEG-rLDP-AE showed significant antitumor activity both in vitro and in vivo . After PEGylation, mPEG-rLDP still retained the binding capability to the enediyne AE and presented the physicochemical characteristics similar to that of native LDP. It is of interest that the PEGylation did not diminish the antitumor efficacy of LDM, implying the possibility that this derivative may function as a payload to deliver novel tumor-targeted drugs.
DOI:10.1016/j.apsb.2015.03.006      URL    
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关键词（key words）
赖氨甘草次酸
增溶
色谱法
高效液相
抗肿瘤活性
Glycyrrhetinic acid lysin...
Solubilization
Chromatography
high performance liquid
Antitumor activity
作者
张丽娟
钟家亮
古同男
魏秋芬
ZHANG Lijuan
ZHONG Jialiang
GU Tongnan
WEI Qiufen