氨基酸单羰基姜黄素缀合物的合成及其在肝细胞癌中的抗肿瘤评估-动脉网

Synthesis and antitumor evaluation of amino acid conjugates of monocarbonyl curcumin in hepatocellular carcinoma cell

Nature 等信源发布 2025-03-10 16:11



可切换为仅中文







Abstract

摘要

Curcumin possesses a variety of pharmacological properties, particularly anticancer activity. However, its clinical utility is limited by its poor water solubility and low bioavailability. To alleviate the problems, our previous research demonstrated that mono-carbonyl curcumin easters can be employed for the development of novel anticancer agents.

姜黄素具有多种药理特性，尤其是抗癌活性。然而，其临床应用受到水溶性差和生物利用度低的限制。为了解决这些问题，我们之前的研究表明，单羰基姜黄素酯可用于开发新型抗癌药物。

In this study, further structural optimization was performed and a series of novel amino acid conjugates of mono-carbonyl curcumin .

在这项研究中，进一步进行了结构优化，并获得了一系列新型的单羰基姜黄素氨基酸共轭物。

H1-H6

were designed, synthesized, and evaluated by in vitro and in vivo studies. Compound

进行了设计、合成，并通过体外和体内研究进行了评估。化合物

一级标题

was found as the most potent derivative (IC

被发现是最有效的衍生物 (IC

= 8.66 µM) compared to curcumin (IC

= 8.66 µM) 与姜黄素 (IC

= 36.19 µM) by anti-proliferation assay. Subsequently, wound healing, transwell, JC-1 staining, the HepG2 cell xenograft model and H&E staining experiments were performed, and it was found that compound

通过抗增殖实验测定，= 36.19 µM。随后进行了伤口愈合、Transwell、JC-1染色、HepG2细胞异种移植模型以及H&E染色实验，发现化合物

一级标题

was more effective than curcumin in inhibiting tumor growth. The results of transcriptome sequence, bioinformatics analysis, molecular docking, and western blotting suggested that compound

在抑制肿瘤生长方面比姜黄素更有效。转录组序列、生物信息学分析、分子对接和蛋白质印迹的结果表明，该化合物

一级标题

could inhibit cell proliferation and induce apoptosis through the AKT/FOXO1 pathway. Hence,

可以通过AKT/FOXO1通路抑制细胞增殖并诱导凋亡。因此，

is a promising lead compound with the potential to be developed as a chemotherapy agent for hepatocellular carcinoma.

是一个有希望的先导化合物，有可能被开发为肝细胞癌的化疗药物。

Introduction

简介

Curcumin is a hydrophobic polyphenol from the rhizome of the plant

姜黄素是一种来自植物根茎的疏水性多酚

Curcuma longa

姜黄

with a broad spectrum of pharmacological activities, especially antitumor activity

具有广泛的药理活性，尤其是抗肿瘤活性

，

. However, clinical investigations using curcumin did not show great benefit due to its poor bioavailability

然而，由于姜黄素的生物利用度较低，使用它进行的临床研究并未显示出显著的效果。

，

. A series of modifications have been performed on the β-diketone structure and hydroxy on benzene of curcumin, which could largely enhance stability, bioavailability, and antitumor activity

对姜黄素的β-二酮结构和苯环上的羟基进行了一系列修饰，这些修饰可以大幅提高其稳定性、生物利用度和抗肿瘤活性。

，

。

Our previous research showed that mono-carbonyl curcumin cinnamyl ester

我们之前的研究表明，单羰基姜黄素肉桂酸酯

and phenylmethyl ester

苯甲基酯

greatly increased anti-human hepatocellular carcinoma (HCC) activity, which converted the diketone structure into a single ketone and modified hydroxy on benzene of curcumin with natural organic acids

显著增强了抗人肝细胞癌（HCC）活性，将双酮结构转化为单酮，并用天然有机酸修饰了姜黄素苯环上的羟基。

，

. The results of molecular dynamics simulations and pharmacological experiments suggested the subsequent structural optimization may focus on shortening the carbon chain modification on hydroxy on benzene suitably and introducing a moiety with better water solubility. The mechanism of curcumin and its derivatives was also simply explored in our previous research and may relate to the influence of the HepG2 cell metabolic process through inhibition of the expression of AKT protein.

分子动力学模拟和药理学实验结果表明，后续的结构优化可以集中于适当缩短苯环上羟基的碳链修饰，并引入水溶性更好的基团。在我们之前的研究中也对姜黄素及其衍生物的作用机制进行了简单探索，可能与通过抑制AKT蛋白的表达影响HepG2细胞代谢过程有关。

However, how AKT regulates downstream signaling molecules remains unclear and it is important to investigate urgently the mechanism for anticancer effects of Traditional Chinese Medicine..

然而，AKT如何调节下游信号分子仍不清楚，迫切需要研究中药抗癌作用的机制。

Amino acids are the primary construction blocks for proteins with biological activities and a broader shift can result in structural modification accounting for amino acids containing a variety of sidechains

氨基酸是具有生物活性的蛋白质的主要构建块，更广泛的转变可能导致结构修饰，从而解释了含有各种侧链的氨基酸。

，

. Among the amino acid and natural compound conjugates, oncolytic peptides-anticancer compounds based conjugates represent promising novel candidates for anticancer treatments

在氨基酸和天然化合物的结合物中，基于溶瘤肽的抗癌化合物结合物代表了有前景的抗癌治疗新候选者。

，

. To date, several amino acid and natural compound conjugates have been reported that involve curcumin, astaxanthin and quercetin to enhance the pharmacological activities, which may be attributed to their better solubility, enhanced accumulation in the cells, resulting from better cellular uptake and decreased metabolic rate.

到目前为止，已经报道了多种氨基酸和天然化合物的结合物，这些结合物涉及姜黄素、虾青素和槲皮素，以增强药理活性，这可能归因于它们更好的溶解性、细胞内积累增加，这是由于更好的细胞摄取和降低的代谢率所致。

，

. These ester linkages are biodegradable by esterase enzymes and therefore these conjugates may be acting as prodrugs

这些酯键可被酯酶降解，因此这些结合物可能充当了前药的作用。

，

. Moreover, the assessment of their antiproliferative activities suggested that diesters of curcumin were relatively more active than curcumin itself and the monoester of curcumin in HeLa and KB cancer cells due to their increased solubility, slow metabolism and better cellular uptake

此外，对其抗增殖活性的评估表明，由于双酯类姜黄素的溶解性增加、代谢缓慢以及细胞摄取更好，在HeLa和KB癌细胞中，它们的活性相对高于姜黄素本身及其单酯形式。

. Therefore, it is a strategy that introduces natural products such as amino acids on the hydroxy of mono-carbonyl curcumin to increase the anti-HCC activity.

因此，它是一种在单羰基姜黄素的羟基上引入氨基酸等天然产物以增加抗肝细胞癌活性的策略。

In this study, six highly water-soluble amino acid conjugates

本研究中，六个高水溶性氨基酸结合物

一级标题

of mono-carbonyl curcumin were synthesized through structural modification of curcumin. The anti-HCC activities of the conjugates were assessed by a series of in vitro and in vivo experiments including MTT assay, clone formation, wound healing, transwell migration, JC-1, Xenograft model, H&E staining analysis, molecular docking, western blot and some informatics analysis.

通过结构改造合成了单羰基姜黄素衍生物。通过一系列体外和体内实验评估了这些化合物的抗肝细胞癌活性，包括MTT实验、克隆形成、伤口愈合、Transwell迁移、JC-1、异种移植模型、H&E染色分析、分子对接、蛋白质印迹和一些信息学分析。

The study findings will establish a groundwork for the application of optimizing curcumin in the field of anticancer drugs..

研究结果将为优化姜黄素在抗癌药物领域的应用奠定基础。

Materials and methods

材料与方法

Chemistry

化学

General

通用

All solvents and reagents were analytical grade and purchased from Shanghai Titan Technology Co., LTD.

所有溶剂和试剂均为分析纯，并购自上海泰坦科技股份有限公司。

H NMR and

核磁共振氢谱和

C NMR spectra for the compounds were recorded with Bruker AM 600 MHz and 400 MHz spectrometers using CDCl

化合物的C NMR谱图使用Bruker AM 600 MHz和400 MHz光谱仪记录，溶剂为CDCl。

solvent. Coupling constants (J values) are given in Hz. Melting points (M.P.) were determined on an SRS OptiMelt-100 instrument and ESI-MS was recorded on an Agilent 6520B Q-TOF system. Reactions were monitored by thin-layer chromatography (TLC) on a glass plate coated with silica gel with the fluorescent indicator (GF254).

溶剂。耦合常数（J 值）以 Hz 为单位给出。熔点（M.P.）使用 SRS OptiMelt-100 仪器测定，ESI-MS 在 Agilent 6520B Q-TOF 系统上记录。反应通过涂有硅胶和荧光指示剂（GF254）的玻璃板进行薄层色谱（TLC）监测。

Column chromatography (CC) was performed on silica gel (200–300 mesh; Qingdao Makall Group Co., Ltd.; Qingdao, China)..

柱层析（CC）在硅胶（200-300目；中国青岛玛卡集团有限公司；青岛，中国）上进行。

Preparation of mono-carbonyl Curcumin derivatives H1–H6

单羰基姜黄素衍生物H1-H6的制备

The starting material mono-carbonyl curcumin

起始物料单羰基姜黄素

(Fig.

（图。

) was synthesized from 4-hydroxy-3-methoxybenzaldehyde by the same procedure as described in our previous article

) 是通过与我们之前文章中描述的相同方法，由4-羟基-3-甲氧基苯甲醛合成的

. Specifically, the review emphasizes the role of D-amino acids and D-type amino acids-based conjugates in evading proteolytic cleavage and enhancing tumor accumulation through stereochemical-driven interactions, and the principles directly informed our design of curcumin-D-alanine conjugates

特别地，本综述强调了D-氨基酸及基于D型氨基酸的共轭物在通过立体化学驱动的相互作用规避蛋白水解切割和增强肿瘤积累方面的作用，这些原则直接指导了我们对姜黄素-D-丙氨酸共轭物的设计。

. Six amino acids (glycine, D-alanine, L-valine, L-leucine, L-methionine, L-phenylalanin) were protected by introducing Boc group to the amino group with Et

六种氨基酸（甘氨酸、D-丙氨酸、L-缬氨酸、L-亮氨酸、L-蛋氨酸、L-苯丙氨酸）通过引入Boc基团对氨基进行保护，使用Et。

N and (Boc)

N 和 (Boc)

O in water and acetone mixture solvents at 0–40 ℃ for 4 h. Boc-amino acids (

在0-40℃的水和丙酮混合溶剂中进行4小时。Boc-氨基酸（

2a-2f

), mono-carbonyl curcumin

），单羰基姜黄素

, 1-(3-dimethylaminopropyl)−3-ethylcarbodiimide (EDCI), 4-dimethylaminopyridine (DMAP) were combined with dichloromethane and allowed to react at 25 ℃ for 7–12 h under inert gas to obtain Boc-amino acids curcumin (

，1-（3-二甲氨基丙基）-3-乙基碳二亚胺（EDCI）、4-二甲氨基吡啶（DMAP）与二氯甲烷混合，在惰性气体下于25℃反应7-12小时，得到Boc-氨基酸姜黄素（

3a-3f

). Subsequently, compounds

). 随后，化合物

3a-3f

were allowed to react with 2 mol/L hydrochloric acid in methanol solution at 0 ℃ for 2–3 h to deprotect the Boc group and produce amino acid conjugates of mono-carbonyl curcumin

在0℃下，与2 mol/L的盐酸甲醇溶液反应2-3小时，以脱除Boc保护基，并生成单羰基姜黄素的氨基酸偶联物。

一级标题

。

Fig. 1

图1

Synthesis of amino acid conjugates of mono-carbonyl curcumin

单羰基姜黄素的氨基酸缀合物的合成

. Reagents and conditions: (a) (BOC)

. 试剂和条件：(a) (BOC)

O, triethylamine, acetone and water, 0–40 ℃, 4 h; (b) EDCI, DMAP, N

O，三乙胺，丙酮和水，0–40 ℃，4小时；(b) EDCI，DMAP，N

protection, CH

保护，CH

氯

, 7–12 h; (c) 2 M HCl in CH

，7-12小时；（c）CH中的2 M HCl

OH solution, 0℃, 2–3 h.

氢氧化物溶液，0℃，2-3小时。

Full size image

全尺寸图像

Biological assays

生物测定

Cell culture

细胞培养

Human hepatocellular carcinoma cell line HepG2 was obtained from Nanjing Forestry University. MTT (3-(4,5-Dimethylthiazol-2-yl)−2,5-diphenyltetrazolium bromide) was purchased from Sigma. 6-well, 24-well and 96-well plates were purchased from Beyotime Biotechnology. Cells were grown in DMEM medium with 10% fetal bovine serum at 37 °C and 5% CO.

人肝癌细胞系HepG2购自南京林业大学。MTT（3-（4,5-二甲基噻唑-2-基）-2,5-二苯基四氮唑溴化物）购自Sigma公司。6孔、24孔和96孔板购自碧云天生物技术公司。细胞在含10%胎牛血清的DMEM培养基中，于37°C和5% CO₂条件下培养。

. All compounds were dissolved in DMSO and the final concentration had no effects on cell viability.

所有化合物均溶解于DMSO中，且最终浓度对细胞活力无影响。

MTT assay

MTT检测

Seed HepG2 cells (5000 cells/well) in a 96-well plate and allow them to adhere. The wells were treated with mono-carbonyl curcumin derivatives

在96孔板中接种HepG2细胞（5000个细胞/孔），并让其贴壁。用单羰基姜黄素衍生物处理孔。

H1-H6

with different concentrations and incubated for 48 h. Then, 20 µL, 4 mg/mL MTT of PBS solution was added to each well, allowing viable cells to reduce MTT to formazan crystals. Add 200 µL of DMSO to dissolve the formazan crystals and measure absorbance at 490 nm to determine cell viability on a Super Microplate Reader (MQX2OO) (BioTek, the United States).

使用不同浓度并孵育48小时。然后，向每个孔中加入20 µL、4 mg/mL的MTT PBS溶液，使活细胞将MTT还原为甲臜晶体。加入200 µL DMSO溶解甲臜晶体，并在490 nm处测量吸光度，以通过超微孔板读数仪（MQX2OO）（BioTek，美国）测定细胞活力。

。

Clone formation assay

克隆形成实验

HepG2 cell line (500 cells per well) was seeded into each well of a 6-well plate. The colonies were formed at 37 °C in a CO₂ incubator for 14 days. Subsequently, the colonies were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet solution for visualization

将HepG2细胞系（每孔500个细胞）接种到6孔板的每个孔中。在37°C的CO₂培养箱中培养14天后形成克隆。随后，用4%多聚甲醛固定克隆，并用0.1%结晶紫溶液染色以进行可视化。

。

Wound healing assay

伤口愈合实验

A monolayer of HepG2 cells (1.5 × 10

一层HepG2细胞（1.5×10

cells/ well) was put into 24-well plates and allowed to grow until it reached 80–90% confluence. Use a sterile pipette tip to create a straight line (wound) across the cell monolayer. The detached cells were rinsed gently with PBS. The cells were subjected to the target compound at different concentrations in fresh DMEM medium and subsequently cultured for 24–48 h.

将细胞/孔接种到24孔板中，并使其生长至80-90%融合度。使用无菌移液器吸头在细胞单层上划出一条直线（伤口）。用PBS轻轻冲洗掉脱落的细胞。将细胞置于含不同浓度目标化合物的新鲜DMEM培养基中，随后继续培养24-48小时。

The cell migration was quantified and the percentage of wound closure over time was calculated by measuring the wound width using a DMIL LED 3000 inverted microscope (Leica, Wetzlar, Germany).

通过使用DMIL LED 3000倒置显微镜（Leica，韦茨拉尔，德国）测量伤口宽度，对细胞迁移进行了量化，并计算了随时间变化的伤口闭合百分比。

。

Transwell migration assay

Transwell迁移实验

The upper and lower chambers were separated by placing transwell inserts in a 24-well plate. HepG2 cells (5 × 10

通过在24孔板中放置Transwell小室，将上下腔室分开。HepG2细胞（5×10

cells/ well) were incubated in the upper chamber of each insert and the target compound was added to the lower chamber to promote cell migration through the pores. After incubation for 48 h, remove the inserts and fix the cells on the upper side of the membrane with 4% paraformaldehyde. Stain the migrated cells using crystal violet and count the stained cells on the lower side of the membrane to quantify migration under a microscope.

细胞/孔）被接种在每个插入物的上室中，并将目标化合物添加到下室以促进细胞通过孔隙迁移。孵育48小时后，移除插入物，并用4%多聚甲醛固定膜上侧的细胞。使用结晶紫染色迁移的细胞，并在显微镜下计数膜下侧的染色细胞以量化迁移。

。

Mitochondrial membrane potential assay

线粒体膜电位测定

HepG2 cells were treated with the target compound in a 6-well plate for 48 h to induce changes in mitochondrial function. The medium was removed and washed with PBS. The cells were added to the JC-1 staining solution and incubated for 20 min at 37 °C. After incubation, the cells were washed twice with PBS to remove excess dye.

HepG2细胞在6孔板中用目标化合物处理48小时以诱导线粒体功能的变化。去除培养基并用PBS洗涤。向细胞中加入JC-1染色液，并在37°C下孵育20分钟。孵育结束后，用PBS洗涤细胞两次以去除多余染料。

The cells were then analyzed using a fluorescence microscope and measured both green (488 nm) and red (550 nm) fluorescence intensities. Calculate the ratio of red to green fluorescence intensity. A higher red/green ratio indicates a healthy mitochondrial membrane potential, while a lower ratio suggests mitochondrial dysfunction.

然后使用荧光显微镜分析细胞，并测量绿色（488 纳米）和红色（550 纳米）荧光强度。计算红色与绿色荧光强度的比值。较高的红/绿比值表明线粒体膜电位健康，而较低的比值则提示线粒体功能障碍。

。

Xenograft model and in vivo study

异种移植模型和体内研究

All animal experiments were conducted with the approval of the Animal Care and Use Committee of Anhui University of Science and Technology (License No: S22023017) and in compliance with the Guide for the Care and Use of Laboratory Animals by the National Institutes of Health. The study is reported in accordance with ARRIVE guidelines (.

所有动物实验均经安徽理工大学动物护理和使用委员会批准（许可证编号：S22023017），并遵循美国国立卫生研究院的《实验室动物护理和使用指南》进行。本研究按照ARRIVE指南进行报告。

https://arriveguidelines.org

). The BALB/c nude mice (female, 6–8 weeks, about 20 g) were purchased from Nanjing Junke Biological Co., LTD. Animal welfare and experimental procedures were kept according to the Guide for Care and Use of Laboratory Animals (National Institutes of Health, the United States) and the system guidance of the Anhui University of Science and Technology Animal Management Committee.

). 购买自南京君科生物有限公司的BALB/c裸鼠（雌性，6-8周，约20克）。动物福利和实验程序遵循《美国国立卫生研究院实验室动物饲养与使用指南》以及安徽理工大学动物管理委员会的系统指导。

Mice were anesthetized using the isoflurane gas inhalation anesthesia method. Briefly, HepG2 cell suspension (2 × 10.

使用异氟烷气体吸入麻醉法对小鼠进行麻醉。简言之，将HepG2细胞悬液（2×10^7个细胞）。

cells in 100 µL) was subcutaneously injected into the right flank of nude mice using a sterile syringe. Monitor the mice post-injection for any signs of distress or complications. Provide analgesics as necessary. Place the mice in a suitable housing environment with proper bedding, food, and water.

细胞悬液（100 µL）使用无菌注射器皮下注射到裸鼠右腹部。注射后监测小鼠是否有任何痛苦或并发症的迹象。必要时提供止痛药。将小鼠安置在适当的饲养环境中，提供合适的垫料、食物和水。

Body weights and tumor size were measured every 3 days. The tumor volume was calculated using the formula: 0.5×L×(W).

每3天测量一次体重和肿瘤大小。使用公式计算肿瘤体积：0.5×L×(W)。

, where L and W are the length and width of the tumor mass, respectively. The mice can be euthanized according to ethical guidelines at predetermined time points or when tumors reach a certain size (100 mm³). The tumors were harvested for further analysis such as H&E staining

，其中L和W分别是肿瘤块的长度和宽度。小鼠可以根据伦理指南在预定的时间点或当肿瘤达到一定大小（100 mm³）时实施安乐死。收获肿瘤以进行进一步的分析，如H&E染色。

. At the endpoint, the mice were euthanized post-modeling using pentobarbital sodium.

在实验终点，使用戊巴比妥钠对小鼠进行安乐死。

H&E staining analysis

H&E染色分析

The tissue slide was deparaffinized by immersion in xylene (2 changes, 10–15 min each), followed by 100% ethanol (2 changes), then 95% and 70% ethanol. After being rinsed in distilled water, stained with hematoxylin (5–10 min), differentiated with 1% HCl and 70% ethanol, immersed in ammonia water, stained with eosin (30 s to 2 min), dehydrated in ethanol and xylene, the slide sample was mounted using a coverslip with mounting medium and dried for a few hours and then observed under a light microscope.

组织切片通过浸入二甲苯（更换两次，每次10-15分钟）进行脱蜡，随后用100%乙醇（更换两次）、95%和70%乙醇处理。在蒸馏水中冲洗后，用苏木精染色（5-10分钟），用1%盐酸和70%乙醇分化，浸入氨水中，再用伊红染色（30秒至2分钟），然后在乙醇和二甲苯中脱水。将切片样本用盖玻片和封片介质封片，干燥数小时后，在光学显微镜下观察。

。

GO and KEGG analysis

GO和KEGG分析

Enrichment analysis of gene ontology (GO) function and Kyoto encyclopedia of genes and genomes (KEGG) pathway was performed using the online David (

使用在线David进行基因本体（GO）功能和京都基因与基因组百科全书（KEGG）通路的富集分析（

https://david.ncifcrf.gov/

) database, and

) 数据库，以及

< 0.05 was used as the evaluation criterion. The top 10 biological functions including cellular components (CC), molecular functions (MF), and biological processes (BP), and the top 30 signaling pathways in the results were selected for visual analysis. The column and bubble graphs analyzed by GO and KEGG analysis were finally also conducted.

<0.05 作为评估标准。选择结果中排名前十的生物功能，包括细胞组分（CC）、分子功能（MF）和生物过程（BP），以及排名前三十的信号通路进行可视化分析。最终还进行了通过GO和KEGG分析生成的柱状图和气泡图。

。

Survival analysis and pathological staging

生存分析与病理分期

The survival analysis and pathological staging of AKT and FOXO1 were carried out by the platform GEPIA2 (

通过平台GEPIA2进行AKT和FOXO1的生存分析和病理分期 (

http://gepia2.cancer-pku.cn/

)

. In the GEPIA2 server, Kaplan-Meier survival curves and box plots of liver cancer stage were constructed to examine the relationship between the hub genes and the overall survival and pathological staging of liver cancer patients.

在GEPIA2服务器中，构建了肝癌阶段的Kaplan-Meier生存曲线和箱线图，以检查枢纽基因与肝癌患者的总体生存率和病理分期之间的关系。

Molecular docking studies

分子对接研究

The molecular docking technique was performed using the Glide module in Schrodinger 2017 to explore the interaction mode between the test compounds and AKT and FOXO1 proteins

使用Schrodinger 2017中的Glide模块进行分子对接技术，以探索测试化合物与AKT和FOXO1蛋白之间的相互作用模式。

. The crystal structure of AKT (PDB ID: 3O96) and FOXO1 (PDB ID: 6AEJ) was downloaded from the Protein Data Bank

从蛋白质数据库下载了AKT（PDB ID：3O96）和FOXO1（PDB ID：6AEJ）的晶体结构。

. The initial structures were prepared with the Protein Preparation Wizard workflow and the receptor grid was generated according to the endogenous ligand. The small molecules were optimized with the LigPrep workflow and minimized using the OPLS-2005 force field with the conformational search method.

初始结构通过蛋白质准备向导工作流程制备，并根据内源性配体生成受体网格。小分子通过LigPrep工作流程进行优化，并使用OPLS-2005力场和构象搜索方法进行最小化。

All other parameters were set as default and the binding affinity was evaluated by the binding free energy (Kcal/mol) with standard precision (SP) docking mode. Finally, plausible docking models were selected from the abundant clusters between the ligand and receptor that had lower binding energies and intermolecular interactions were illustrated using PyMOL software..

所有其他参数均为默认设置，结合亲和力通过标准精度（SP）对接模式下的结合自由能（Kcal/mol）进行评估。最后，从配体和受体之间丰富的簇中选择合理的对接模型，这些模型具有较低的结合能，并使用PyMOL软件展示了分子间相互作用。

Western blot analysis

西方墨点法分析

HepG2 cells in 6-well plates were rinsed twice with cold PBS and lysed on ice for 30 min. After centrifugation, the protein concentrations were measured using the BCATM protein quantification kit (Beyotime Biotechnology). Antibodies against phospho-AKT (D9E), FOXO1 (C29H4), p27 Kip1 (D69C12), phospho-Bcl2 (5H2), cleaved-caspase 3 (5AE1) and β-actin (13E5) were purchased from CST.

HepG2细胞在6孔板中用冷PBS洗涤两次，并在冰上裂解30分钟。离心后，使用BCATM蛋白定量试剂盒（Beyotime Biotechnology）测量蛋白浓度。抗磷-AKT（D9E）、FOXO1（C29H4）、p27 Kip1（D69C12）、磷-Bcl2（5H2）、切割-caspase 3（5AE1）和β-肌动蛋白（13E5）的抗体购自CST。

Briefly, the quantified protein samples were subjected to an SDS-PAGE and then transferred to a PVDF membrane. After incubating with primary antibodies at 4 °C overnight and secondary antibodies at room temperature for 2 h, the membrane was illuminated with ECL solution and the density was analyzed using Image J.

简而言之，将定量的蛋白质样品进行SDS-PAGE，然后转移到PVDF膜上。在4°C下与一抗孵育过夜，并在室温下与二抗孵育2小时后，用ECL溶液显影膜，并使用Image J分析密度。

。

Results and discussion

结果与讨论

General method for synthesis of Boc-amino acids 2a-2f

合成Boc-氨基酸2a-2f的通用方法

Six amino acids (20 mmol) were dissolved with 20 mL water and 40 mL acetone in the flask, Et

六种氨基酸（20 mmol）在烧瓶中用20 mL水和40 mL丙酮溶解，Et

N (1.5 equivalent) and (Boc)

N (1.5 当量) 和 (Boc)

O (22 mmol) were added under agitation at a controlled temperature of 0–40 ℃ for 4 h. The reaction mixture was monitored using TLC and the acetone was evaporated under reduced pressure. Then the water layer was acidized with dilute HCl to 2–3 (pH value) and was extracted with ethyl acetate (4 × 60 mL).

在0-40℃的受控温度下，搅拌加入O（22 mmol），持续4小时。使用TLC监测反应混合物，并在减压下蒸发丙酮。然后用水稀盐酸将水层酸化至pH值2-3，并用乙酸乙酯（4×60 mL）进行萃取。

The combined organic layer was then washed with saturated salt (2 × 10 mL), and dried over anhydrous sodium sulfate. The obtained crude product was crystallized with ethyl acetate and petroleum ether (1: 2, volume ratio) and relevant Boc-amino acids .

合并后的有机层用饱和盐水（2×10 mL）洗涤，并用无水硫酸钠干燥。所得粗产物用乙酸乙酯和石油醚（1:2，体积比）结晶，并与相关的Boc-氨基酸处理。

2a-2f

were obtained (90–95% yield).

获得了（90-95%的产率）。

Boc-Glycine (

Boc-甘氨酸 (

)

Yield: 93%. ESI-MS

产量：93%。ESI-MS

米

(M-H)

−

: calculated 174.08, measured 174.18. M.P.:86–88 °C.

：计算值174.08，实测值174.18。熔点：86–88°C。

H NMR (600 MHz, CDCl

1H NMR (600 MHz, CDCl3)

) δ 11.30 (s, 1H), 3.92 (d,

= 40.8 Hz, 2 H), 1.41 (s, 9 H).

C NMR (151 MHz, CDCl

) δ 174.64, 156.22, 81.94, 80.50, 43.42, 42.26, 28.35.

) δ 174.64, 156.22, 81.94, 80.50, 43.42, 42.26, 28.35。

Boc-D-Alanine (

Boc-D-丙氨酸 (

)

Yield: 93%. ESI-MS

产量：93%。ESI-MS

米

(M-H)

−

: calculated 188.10, measured 188.21. M.P.:81–83 °C.

：计算值188.10，实测值188.21。熔点：81–83°C。

H NMR (600 MHz, CDCl

核磁共振氢谱 (600 MHz, 氯仿-d)

) δ 7.92 (s, 1H), 4.23 (d,

= 112.4 Hz, 1H), 1.42 (s, 9 H), 1.40 (d,

= 7.2 Hz, 3 H).

= 7.2 Hz, 3 H)。

C NMR (151 MHz, CDCl

C NMR（151 MHz，CDCl

) δ 177.58, 155.62, 81.74, 80.37, 50.34, 49.21, 28.40, 18.45.

) δ 177.58、155.62、81.74、80.37、50.34、49.21、28.40、18.45。

Boc- L-Valine (

Boc-L-缬氨酸 (

)

Yield: 90%. ESI-MS

产量：90%。ESI-MS

米

(M-H)

−

: calculated 216.13, measured 216.26. M.P.:77–79 °C.

：计算值216.13，实测值216.26。熔点：77–79°C。

H NMR (600 MHz, CDCl

) δ 11.66 (s, 1H), 4.12 (d,

= 138.7 Hz, 1H), 2.33–2.00 (m, 1H), 1.43 (s, 9 H), 0.94 (dd,

= 39.8, 6.8 Hz, 6 H).

C NMR (151 MHz, CDCl

) δ 177.29, 155.99, 81.76, 80.17, 60.21, 58.54, 31.19, 28.42, 19.12, 17.56.

) δ 177.29、155.99、81.76、80.17、60.21、58.54、31.19、28.42、19.12、17.56。

Boc-L-Leucine (

Boc-L-亮氨酸 (

二维

)

Yield: 92%. ESI-MS

产量：92%。ESI-MS

米

(M-H)

−

: calculated 230.15, measured 230.29. M.P.:85–87 °C.

：计算值230.15，测量值230.29。熔点：85–87°C。

H NMR (600 MHz, CDCl

) δ 6.88 (s, 1H), 4.83 (d,

= 164.2 Hz, 1H), 4.21 (d,

= 104.1 Hz, 1H), 1.43 (s, 9 H), 0.94 (d,

= 6.6 Hz, 6 H).

C NMR (151 MHz, CDCl

) δ 178.01, 155.86, 81.72, 80.31, 53.33, 52.14, 41.56, 28.43, 24.92, 22.96, 21.90.

) δ 178.01、155.86、81.72、80.31、53.33、52.14、41.56、28.43、24.92、22.96、21.90。

Boc-L-methionine (

Boc-L-甲硫氨酸 (

)

Yield: 95%. ESI-MS

产量：95%。ESI-MS

(M-H)

−

: calculated 248.10, measured 248.33. M.P.:47–49 °C.

：计算值248.10，实测值248.33。熔点：47–49°C。

H NMR (600 MHz, CDCl

1H NMR (600 MHz, CDCl3)

) δ 10.29 (s, 1H), 4.51–4.21 (m, 1H), 2.56 (t,

= 7.1 Hz, 2 H), 2.22–2.12 (m, 1H), 2.09 (s, 3 H), 2.02–1.92 (m, 1H), 1.43 (s, 9 H).

= 7.1 Hz, 2 H), 2.22–2.12 (m, 1H), 2.09 (s, 3 H), 2.02–1.92 (m, 1H), 1.43 (s, 9 H)。

C NMR (151 MHz, CDCl

) δ 176.96, 155.74, 82.18, 80.51, 53.63, 52.80, 31.93, 30.07, 28.39, 15.49.

) δ 176.96、155.74、82.18、80.51、53.63、52.80、31.93、30.07、28.39、15.49。

Boc-L-Phenylalanine (

Boc-L-苯丙氨酸 (

)

Yield: 94%. ESI-MS

产量：94%。ESI-MS

米

(M-H)

−

: calculated 264.13, measured 264.31. M.P.:85–87 °C.

：计算值264.13，实测值264.31。熔点：85–87°C。

H NMR (600 MHz, CDCl

) δ 11.77 (s, 1H), 7.34–7.27 (m, 3 H), 7.22 (dd,

= 15.7, 7.1 Hz, 2 H), 4.54 (d,

= 145.3 Hz, 1H), 3.29–2.87 (m, 2 H), 1.38 (d,

= 80.2 Hz, 9 H).

= 80.2 Hz, 9 H)。

C NMR (151 MHz, CDCl

) δ 176.73, 155.47, 136.63, 135.98, 129.51, 128.67, 127.18, 81.71, 80.38, 56.22, 54.37, 39.32, 37.94, 28.40.

) δ 176.73, 155.47, 136.63, 135.98, 129.51, 128.67, 127.18, 81.71, 80.38, 56.22, 54.37, 39.32, 37.94, 28.40。

General method for synthesis of mono-carbonyl curcumin Boc-amino acid conjugates 3a-3f

合成单羰基姜黄素Boc-氨基酸缀合物3a-3f的通用方法

A solution of mono-carbonyl curcumin

单羰基姜黄素溶液

(1 g, 3.1 mmol) and Boc-amino acids (7.5 mmol) in CH

(1 g, 3.1 mmol) 和 Boc-氨基酸 (7.5 mmol) 在 CH 中

氯

(25 ml) was taken in a round-bottomed flask. DMAP (1.5 mmol) was added to the reaction mixture and stirred for 0.5 h. After this EDCI (7.5 mmol) was added and set for stirring until the completion of the reaction (7–12 h) under a nitrogen atmosphere. The progress of the reaction was monitored by TLC analysis.

(25毫升)圆底烧瓶中取样，加入DMAP（1.5毫摩尔）到反应混合物中并搅拌0.5小时。之后加入EDCI（7.5毫摩尔），在氮气氛围下搅拌至反应完成（7-12小时）。通过TLC分析监测反应进程。

The reaction mixture was then added H.

然后向反应混合物中加入 H。

O and extracted with CH

O 并用 CH 萃取

氯

. The combined organic layer was dried over anhydrous sodium sulfate and was purified by flash chromatography with ethyl acetate/petroleum ether to give these yellow compounds

合并的有机层用无水硫酸钠干燥，并通过快速柱层析（乙酸乙酯/石油醚）纯化，得到这些黄色化合物。

. The yields were between 51.3% and 61.5%.

产量在51.3%到61.5%之间。

((1E,4E)−3-oxopenta-1,4-diene-1,5-diyl)bis(2-methoxy-4,1-phenylene) bis(2-((tert-butoxycarbonyl)amino)acetate) (

((1E,4E)-3-氧代戊-1,4-二烯-1,5-二基)双(2-甲氧基-4,1-亚苯基) 双(2-((叔丁氧基羰基)氨基)乙酸酯) (

)

Yield: 60.8%. ESI-MS

产率：60.8%。ESI-MS

米

: 639.69 (M-H)

：639.69（M-H）

−

。

H NMR (600 MHz, CDCl

1H NMR (600 MHz, CDCl3)

) δ 7.45 (d,

= 16.2 Hz, 1H), 7.15–7.05 (m, 3 H), 6.65 (d,

= 16.2 Hz, 1H), 4.21 (d,

= 5.5 Hz, 1H), 3.83 (s, 3 H), 2.37 (s, 2 H), 1.45 (s, 9 H).

C NMR (151 MHz, CDCl

) δ 198.35, 168.57, 155.78, 151.39, 142.68, 141.25, 133.77, 127.56, 123.29, 121.59, 111.43, 80.32, 56.02, 42.39, 28.41, 27.63.

) δ 198.35, 168.57, 155.78, 151.39, 142.68, 141.25, 133.77, 127.56, 123.29, 121.59, 111.43, 80.32, 56.02, 42.39, 28.41, 27.63。

((1E,4E)−3-oxopenta-1,4-diene-1,5-diyl)bis(2-methoxy-4,1-phenylene) bis(2-((tert-butoxycarbonyl)amino)propanoate) (

((1E,4E)-3-氧代戊-1,4-二烯-1,5-二基)双(2-甲氧基-4,1-亚苯基) 双(2-((叔丁氧羰基)氨基)丙酸酯) (

)

Yield: 55.8%. ESI-MS

产率：55.8%。ESI-MS

米

: 667.74 (M-H)

：667.74（M-H）

−

。

H NMR (600 MHz, CDCl

) δ 7.47 (d,

= 16.2 Hz, 1H), 7.13 (q,

= 11.3, 10.4 Hz, 3 H), 6.66 (d,

= 16.2 Hz, 1H), 5.09 (s, 1H), 4.60 (s, 1H), 3.85 (s, 3 H), 2.39 (s, 3 H), 1.46 (s, 9 H).

C NMR (151 MHz, CDCl

) δ 198.33, 183.23, 155.23, 151.47, 142.74, 141.53, 133.74, 127.59, 123.34, 121.68, 111.43, 56.09, 49.46, 29.86, 28.48, 27.70, 18.88.

) δ 198.33, 183.23, 155.23, 151.47, 142.74, 141.53, 133.74, 127.59, 123.34, 121.68, 111.43, 56.09, 49.46, 29.86, 28.48, 27.70, 18.88。

((1E,4E)−3-oxopenta-1,4-diene-1,5-diyl)bis(2-methoxy-4,1-phenylene) bis(2-((tert-butoxycarbonyl)amino)−3-methylbutanoate) (

((1E,4E)-3-氧代戊-1,4-二烯-1,5-二基)双(2-甲氧基-4,1-亚苯基) 双(2-((叔丁氧羰基)氨基)-3-甲基丁酸酯) (

)

Yield: 53.4%. ESI-MS

产率：53.4%。ESI-MS

米

: 723.85 (M-H)

−

。

H NMR (600 MHz, CDCl

1H NMR (600 MHz, CDCl3)

) δ 7.46 (d,

= 16.2 Hz, 1H), 7.14–7.11 (m, 2 H), 7.07 (d,

= 8.0 Hz, 1H), 6.66 (d,

= 16.2 Hz, 1H), 5.09 (s, 1H), 4.52 (s, 1H), 3.83 (s, 3 H), 1.46 (s, 9 H), 1.09 (d,

= 6.8 Hz, 3 H), 1.03 (d,

= 6.8 Hz，3 H），1.03（d，

= 6.8 Hz, 3 H).

= 6.8 Hz, 3 H)。

C NMR (151 MHz, CDCl

C NMR（151 MHz，CDCl

) δ 198.34, 170.47, 155.84, 151.47, 142.74, 141.43, 133.73, 127.55, 123.44, 121.64, 111.38, 80.09, 58.69, 55.86, 31.43, 28.45, 27.66, 19.22, 17.34.

) δ 198.34, 170.47, 155.84, 151.47, 142.74, 141.43, 133.73, 127.55, 123.44, 121.64, 111.38, 80.09, 58.69, 55.86, 31.43, 28.45, 27.66, 19.22, 17.34。

((1E,4E)−3-oxopenta-1,4-diene-1,5-diyl)bis(2-methoxy-4,1-phenylene) bis(2-((tert-butoxycarbonyl)amino)−4-methylpentanoate) (

((1E,4E)-3-氧代戊-1,4-二烯-1,5-二基)双(2-甲氧基-4,1-亚苯基) 双(2-((叔丁氧羰基)氨基)-4-甲基戊酸酯) (

3天

)

Yield: 51.3%. ESI-MS

产率：51.3%。ESI-MS

米

: 751.90 (M-H)

：751.90（M-H）

−

。

H NMR (600 MHz, CDCl

1H NMR (600 MHz, CDCl3)

) δ 7.45 (d,

= 16.2 Hz, 1H), 7.10 (t,

= 13.0 Hz, 3 H), 6.64 (d,

= 16.2 Hz, 1H), 4.99 (s, 1H), 4.56 (s, 1H), 3.82 (s, 3 H), 1.83 (d,

= 19.9 Hz, 2 H), 1.67–1.60 (m, 1H), 1.45 (s, 9 H), 1.00 (d,

= 5.8 Hz, 6 H).

= 5.8 Hz, 6 H)。

C NMR (151 MHz, CDCl

C NMR（151 MHz，CDCl

) δ 198.29, 171.52, 155.51, 151.46, 142.74, 141.55, 133.61, 127.46, 123.35, 121.61, 111.39, 80.06, 55.96, 52.34, 41.76, 28.41, 27.61, 24.90, 23.02, 21.99.

) δ 198.29, 171.52, 155.51, 151.46, 142.74, 141.55, 133.61, 127.46, 123.35, 121.61, 111.39, 80.06, 55.96, 52.34, 41.76, 28.41, 27.61, 24.90, 23.02, 21.99。

((1E,4E)−3-oxopenta-1,4-diene-1,5-diyl)bis(2-methoxy-4,1-phenylene) bis(2-((tert-butoxycarbonyl)amino)−4-(methylthio)butanoate) (

((1E,4E)-3-氧代戊-1,4-二烯-1,5-二基)双(2-甲氧基-4,1-亚苯基) 双(2-((叔丁氧羰基)氨基)-4-(甲硫基)丁酸酯) (

)

Yield: 61.5%. ESI-MS

产量：61.5%。ESI-MS

米

: 787.97 (M-H)

−

。

H NMR (600 MHz, CDCl

1H NMR (600 MHz, CDCl3)

) δ 7.46–7.42 (m, 1H), 7.12–7.02 (m, 3 H), 6.64 (d,

= 16.2 Hz, 1H), 5.28 (s, 1H), 4.69 (s, 1H), 3.82 (s, 3 H), 2.34 (d,

= 13.5 Hz, 4 H), 2.12 (s, 3 H), 1.44 (s, 9 H).

C NMR (151 MHz, CDCl

) δ 198.26, 170.43, 151.26, 142.60, 141.25, 133.74, 127.52, 124.96, 123.54, 121.57, 114.98, 111.33, 109.51, 80.24, 55.91, 53.01, 29.87, 28.36, 27.59, 15.51.

) δ 198.26, 170.43, 151.26, 142.60, 141.25, 133.74, 127.52, 124.96, 123.54, 121.57, 114.98, 111.33, 109.51, 80.24, 55.91, 53.01, 29.87, 28.36, 27.59, 15.51。

((1E,4E)−3-oxopenta-1,4-diene-1,5-diyl)bis(2-methoxy-4,1-phenylene) bis(2-((tert-butoxycarbonyl)amino)−3-phenylpropanoate) (

((1E,4E)-3-氧代戊-1,4-二烯-1,5-二基)双(2-甲氧基-4,1-亚苯基) 双(2-((叔丁氧羰基)氨基)-3-苯基丙酸酯) (

)

Yield: 58.2%. ESI-MS

产量：58.2%。ESI-MS

米

: 819.94 (M-H)

：819.94（M-H）

−

。

H NMR (600 MHz, CDCl

1H NMR (600 MHz, CDCl3)

) δ 7.46 (d,

= 16.2 Hz, 1H), 7.37–7.31 (m, 2 H), 7.28 (d,

= 7.1 Hz, 3 H), 7.13 (d,

= 6.5 Hz, 2 H), 7.01 (d,

= 8.3 Hz, 1H), 6.66 (d,

= 16.2 Hz, 1H), 5.02 (d,

= 8.1 Hz, 1H), 4.87 (d,

= 7.0 Hz, 1H), 3.85 (s, 3 H), 3.33 (dd,

= 13.9, 5.5 Hz, 1H), 3.21 (dd,

= 13.9, 6.5 Hz, 1H), 1.42 (s, 9 H).

C NMR (151 MHz, CDCl

) δ 198.27, 170.09, 155.20, 151.46, 142.66, 141.33, 135.99, 133.74, 129.66, 128.71, 127.57, 127.25, 123.37, 121.59, 111.43, 80.19, 55.96, 54.50, 38.26, 28.40, 27.67.

) δ 198.27, 170.09, 155.20, 151.46, 142.66, 141.33, 135.99, 133.74, 129.66, 128.71, 127.57, 127.25, 123.37, 121.59, 111.43, 80.19, 55.96, 54.50, 38.26, 28.40, 27.67。

General method for synthesis of mono-carbonyl curcumin amino acid conjugates H1-H6

合成单羰基姜黄素氨基酸偶联物H1-H6的通用方法

Compounds

化合物

(2 mmol) were allowed to react with 15 mL of 2 mol/L hydrochloric acid methanol solution at 0 °C for 2–3 h, and TLC was used to monitor the reaction progress. After reaction completion, the product was filtered, dried, and purified by recrystallization using ethanol and petroleum ether. This process yielded target compounds .

（2 mmol）在0°C下与15 mL的2 mol/L盐酸甲醇溶液反应2-3小时，并用TLC监测反应进程。反应完成后，过滤产物，干燥并使用乙醇和石油醚重结晶纯化。该过程得到了目标化合物。

H1-H6

with 72–78% yields. (

收率为72-78%。(

) Yield: 77%. ESI-MS

产率：77%。ESI-MS

米

: 439.45 (M-H)

−

. (

。(

) Yield: 73%. ESI-MS

) 产率：73%。ESI-MS

米

: 467.51 (M-H)

−

. (

。（

) Yield: 72%. ESI-MS

产率：72%。ESI-MS

米

: 523.61 (M-H)

−

. (

。(

) Yield: 72%. ESI-MS

产率：72%。ESI-MS

米

: 551.67 (M-H)

−

. (

。(

) Yield: 78%. ESI-MS

产率：78%。ESI-MS

米

: 587.73 (M-H)

−

. (

。(

) Yield: 75%. ESI-MS

产率：75%。ESI-MS

米

: 619.70 (M-H)

−

。

Inhibition effect on cell proliferation in HepG2 cells

对HepG2细胞增殖的抑制作用

The cell proliferation activity of curcumin derivatives

姜黄素衍生物的细胞增殖活性

H1-H6

towards HepG2 cells was evaluated via MTT assay and clone formation assay. As shown in Fig.

对HepG2细胞的作用通过MTT实验和克隆形成实验进行了评估。如图所示。

A, the cell monolayer was incubated with six conjugates of mono-carbonyl curcumin with amino acids

A，细胞单层与六种单羰基姜黄素与氨基酸的结合物一起孵育。

H1-H6

at the concentration of 10

在浓度为10时

微符号 (µ)

M for 48 h. Curcumin and sorafenib were used as positive controls. It was obvious that all of these conjugates except

M持续48小时。姜黄素和索拉非尼作为阳性对照。很明显，除了所有这些结合物之外

exhibited more potent toxicity in HepG2 cells when compared with the blank control (only cells sap added group) and curcumin group. Remarkably, derivative

在HepG2细胞中，与空白对照组（仅添加细胞液的组）和姜黄素组相比，表现出更强的毒性。值得注意的是，衍生物

一级标题

was the most active compound and showed equivalent activity to the positive drug sorafenib, which was chosen for further studies. As shown in Fig.

是最活跃的化合物，显示出与阳性药物索拉非尼相当的活性，因此被选中进行进一步研究。如图所示。

B, the cells were treated with

B，细胞经处理后

一级标题

at different concentrations (0.5, 1, 2, 4, 8, 16, 32, 64 µM) for 48 h. The IC

不同浓度（0.5、1、2、4、8、16、32、64 µM）下处理48小时。IC

values of

值

and curcumin were 8.66 and 36.19 µM, respectively and the anti-proliferation activity of

分别为8.66和36.19 µM，且抗增殖活性为

一级标题

was about 4.18 times higher than that of curcumin. As shown in Fig.

约为姜黄素的4.18倍。如图所示。

C, the clone formation assay also displayed that

C，克隆形成实验也显示了

一级标题

(4 µM) inhibited the clone formation ability of HepG2 cells for 48 h. The results of MTT and clone formation assay indicated that

(4 µM) 抑制了 HepG2 细胞 48 小时的克隆形成能力。MTT 和克隆形成实验的结果表明，

一级标题

exhibited promising anti-proliferation activity in HepG2 cells.

在HepG2细胞中表现出良好的抗增殖活性。

Fig. 2

图2

Anti-proliferation assay to HepG2 cells in vitro. (A) Curcumin derivatives

体外对HepG2细胞的抗增殖实验。（A）姜黄素衍生物

H1-H6

at the concentration of 10

在浓度为10时

微

M for 48 h compared with curcumin and sorafenib. (B)

M 持续 48 小时，与姜黄素和索拉非尼相比。(B)

一级标题

and curcumin at different concentrations (0.5, 1, 2, 4, 8, 16, 32, 64 µM) for 48 h. (C) Clone formation assay to HepG2 cells treatment with

不同浓度（0.5、1、2、4、8、16、32、64 µM）的姜黄素处理48小时。(C) 克隆形成实验用于HepG2细胞处理

(4 µM) for 2 weeks. Data was represented by the mean ± SD of the three independent experiments. **

(4 µM)持续2周。数据以三次独立实验的平均值±标准差表示。**

< 0.01, compared with the curcumin group.

<0.01，与姜黄素组相比。

Full size image

全尺寸图像

Inhibition migration and apoptosis-inducing effects

抑制迁移和诱导凋亡效果

To verify whether derivative

为了验证导数是否

has the anti-migration effect, a wound healing test and transwell migration assay were applied to the test. The results of the wound healing assay in Fig.

具有抗迁移作用，应用伤口愈合实验和Transwell迁移实验进行测试。图中伤口愈合实验的结果。

A indicated that after treating HepG2 cells with 4 µM

A 表示用 4 µM 处理 HepG2 细胞后

一级标题

for 24 h and 48 h, the relative migration distance of cells was markedly reduced in a time-dependent manner compared to the control cells and curcumin. The results of the transwell migration assay in Fig.

在24小时和48小时时，细胞的相对迁移距离与对照组细胞和姜黄素相比，呈现出显著的时间依赖性减少。图中Transwell迁移实验的结果显示。

B showed that the number of traveling through the filter was significantly decreased with

B 表明通过过滤器的行进数量显著减少，

(4 µM) for 48 h. The results of the wound healing test and transwell migration assay together suggested that

(4 µM) 处理 48 小时。伤口愈合实验和 Transwell 迁移实验的结果共同表明，

exhibited the strong potential to suppress HepG2 cell migration and hence inhibit tumor progression.

表现出强烈的抑制HepG2细胞迁移的潜力，从而抑制肿瘤进展。

We next performed a metachromatic fluorochrome JC-1 staining assay to confirm whether compound

我们接下来进行了异色荧光染料JC-1染色实验，以确认化合物是否

一级标题

can induce apoptosis. As shown in Fig.

可以诱导细胞凋亡。如图所示。

C, HepG2 cells were treated with compound

C，HepG2细胞用化合物处理

(4 µM) for 48 h and then stained with JC-1 for 20 min at 37 °C. Both the loss of red fluorescence and the gain of green-emitting monomers indicated that compound

（4 µM）处理48小时，然后在37°C下用JC-1染色20分钟。红色荧光的丧失和绿色单体的增加表明化合物

一级标题

induced apoptosis in the HepG2 cell via depolarizing mitochondrial transmembrane potential.

通过去极化线粒体跨膜电位诱导HepG2细胞凋亡。

Fig. 3

图 3

Wound healing assay (A) and transwell migration assay (B) to HepG2 cells treatment with DMEM medium containing the indicated concentrations (4 µM) of

伤口愈合实验（A）和Transwell迁移实验（B）用于处理含有指定浓度（4 µM）的DMEM培养基的HepG2细胞

一级标题

for 24 h and 48 h. (C) Analysis of apoptotic HepG2 cells by JC-1 staining assay. The cell was treated with

持续24小时和48小时。（C）通过JC-1染色实验分析HepG2细胞的凋亡情况。细胞经处理后，

一级标题

(4 µM) for 48 h, and then stained with JC-1 for 20 min at 37 ℃. The representative images were shown of the corresponding fluorescent channel (40×magnification). Data was represented by the mean ± SD of the three independent experiments. ***

(4 µM)处理48小时，然后在37℃下用JC-1染色20分钟。展示了相应荧光通道的代表性图像（40×放大倍数）。数据以三次独立实验的平均值±标准差表示。***

< 0.001, **

< 0.01, compared with the control group.

< 0.01，与对照组相比。

Full size image

全尺寸图像

Antitumor activity in vivo

体内抗肿瘤活性

Due to the remarkable anti-proliferative, anti-migration and apoptosis induction activities of

由于其显著的抗增殖、抗迁移和诱导凋亡活性

一级标题

in vitro, its anticancer activity in vivo was assessed utilizing a mouse hepatic carcinoma cancer xenograft model established by the subcutaneous inoculation of HepG2 cells. When the tumor volumes reached approximately 100 mm

在体外，其抗癌活性在体内通过皮下接种HepG2细胞建立的小鼠肝癌异种移植模型进行评估。当肿瘤体积达到约100立方毫米时。

, the mice were randomized into 3 groups with 6 mice in each group. Taking an injection of saline as the solvent vehicle control, curcumin (5 mg/kg) and

，将小鼠随机分为3组，每组6只小鼠。以注射生理盐水作为溶剂载体对照，姜黄素（5毫克/千克）和

(5 mg/kg) of 200 µL were injected into mice intraperitoneally administered every day for 27 days. The body weight (Fig.

(5毫克/千克)的200微升被每天腹腔注射到小鼠体内，持续27天。体重（图。

A) and tumor volume (Fig.

A) 和肿瘤体积 (图。

B) of the random selection of nude mice were recorded every three days. During treatment, all the animals in different treatment groups were kept at relatively stable body weights, suggesting the low toxicity of

B) 记录了随机选择的裸鼠每三天的数据。在治疗期间，不同治疗组的所有动物都保持了相对稳定的体重，这表明其毒性较低。

一级标题

. When the tumors were dissected and weighed after compound treatment, the inhibition rate of the average tumor volume of

当肿瘤在化合物处理后被解剖和称重时，平均肿瘤体积的抑制率为

一级标题

-treated mice was significantly better than curcumin. Then, H&E staining of rat liver cells was conducted to observe the difference in cell morphology in different groups of tumor tissues. As illustrated in Fig.

-处理的小鼠显著优于姜黄素。然后，对大鼠肝细胞进行H&E染色，以观察不同组肿瘤组织中细胞形态的差异。如图所示。

C, the results showed that curcumin derivative

C，结果显示姜黄素衍生物

一级标题

destroyed the cell structure of transplanted tumor tissues, resulting in the blue-stained nucleus disappearing and the cells and tissues necrotizing. Overall, these findings indicated that curcumin derivative

破坏了移植瘤组织的细胞结构，导致蓝染的细胞核消失，细胞和组织坏死。总体而言，这些发现表明姜黄素衍生物

effectively inhibited cancer growth in vivo and represented a promising drug candidate for hepatocellular carcinoma.

有效抑制了体内癌症生长，是肝细胞癌的一种有前景的药物候选物。

Fig. 4

图4

Derivative

导数

一级标题

inhibited HepG2 xenograft growth in vivo compared with control and curcumin. Body weight (A) and tumor volume (B) changes of mice were examined every 3 days for 27 days during treatment. Visible tumor formation and photographs of representative tumors removed from mice after treatment. (C) The xenograft tumor tissues of nude mice were observed and photographed through H&E staining (200×).

与对照组和姜黄素相比，抑制了HepG2异种移植瘤在体内的生长。每3天检查一次小鼠的体重（A）和肿瘤体积（B）变化，持续27天。治疗后可见肿瘤形成，并拍摄从小鼠身上取出的代表性肿瘤的照片。（C）通过H&E染色观察并拍摄裸鼠异种移植瘤组织（200×）。

Data was represented by the mean ± SD of the three independent experiments..

数据以三次独立实验的平均值±标准差表示。

Full size image

全尺寸图像

Curcumin-Induced comprehensive transcriptomes profile in HepG2 cells

姜黄素诱导的HepG2细胞全转录组图谱

To explore the underlying mechanism of curcumin, we drew a comprehensive transcriptome sequencing technology and data analysis mining of HepG2 liver cancer cells in Fig.

为了探索姜黄素的潜在机制，我们绘制了图中所示的HepG2肝癌细胞的综合转录组测序技术和数据分析挖掘。

. We found that a total of 5793 differentially expressed genes (DEGs) were significantly up-regulated and 2295 genes down-regulated in the curcumin intervention group compared with the control group. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis were applied to explore the role of DEGs under curcumin treatment..

我们发现，与对照组相比，姜黄素干预组共有5793个差异表达基因（DEGs）显著上调，2295个基因下调。应用基因本体（GO）和京都基因与基因组百科全书（KEGG）富集分析，探讨了姜黄素处理下差异表达基因的作用。

The results showed that in the GO database, the DEGs after curcumin administration were mainly enriched in the regulation of transcription of DNA or RNA, transcription factor activity, DNA replication, intracellular signal transduction, response to extracellular stimuli and regulation of apoptosis, suggesting that curcumin can regulate intracellular signal transduction and the transcription factor activity to affect cell cycle and cell proliferation.

结果表明，在GO数据库中，姜黄素干预后的差异表达基因主要富集在DNA或RNA转录的调控、转录因子活性、DNA复制、细胞内信号转导、对细胞外刺激的反应以及细胞凋亡的调控等，提示姜黄素可以通过调节细胞内信号转导和转录因子活性，影响细胞周期和细胞增殖。

In the KEGG database, the DEGs after drug administration were mainly focused on the PI3K/AKT signaling pathway, FOXO signaling pathway, transcriptional misregulation in cancers, Rap1 signaling pathway, p53 signaling pathway, MAPK signaling pathway, cAMP signaling pathway. It is suggested that curcumin may regulate multiple signaling pathways and affect cell cycle and proliferation after administration..

在KEGG数据库中，给药后的差异表达基因主要集中在PI3K/AKT信号通路、FOXO信号通路、癌症中的转录失调、Rap1信号通路、p53信号通路、MAPK信号通路、cAMP信号通路。这提示姜黄素给药后可能通过调控多条信号通路，影响细胞周期和增殖。

The results of GO and KEGG both indicated that the AKT signaling pathway and FOXO pathway might be involved in the regulation mechanism of curcumin against hepatocellular carcinoma HepG2, providing new ideas for clinical treatment and new drug development.

GO和KEGG的结果均提示AKT信号通路和FOXO通路可能参与了姜黄素抗肝癌HepG2的作用机制，为临床治疗和新药开发提供了新的思路。

Fig. 5

图5

Transcriptome alterations of HepG2 cells were uncovered by high-throughput sequencing. (A) Scatter plot of repeat correlation check. The horizontal and vertical axes are the log

通过高通量测序揭示了HepG2细胞的转录组变化。(A) 重复相关性检查的散点图。横轴和纵轴是log值。

(TPM) values of the two samples respectively. The more similar the samples, the closer the similarity index is to 1, and most of the points in the figure will be clustered near the diagonal. (B) MA map of comparison group expression difference. The horizontal axis is the average log

(TPM)值分别对应两个样本。样品越相似，相似性指数越接近1，图中大部分点会聚集在对角线附近。(B) 比较组表达差异的MA图。横轴为平均log值。

(TPM) of the two groups of samples, that is, (log

两组样本的TPM，即（log

(A) + log

(B))/2, and the vertical axis is log

(B))/2，垂直轴是log

(Foldchange), that is, the log

（倍数变化），即对数

(B/A) value. The red, green, and black spots showed up-regulated, down-regulated, and no difference genes, respectively. (C) GO enrichment analysis. (D) KEGG enrichment analysis of the top 30 DEGs. (E) The FOXO signal pathway.

(B/A) 值。红色、绿色和黑色斑点分别表示上调、下调和无差异基因。(C) GO 富集分析。(D) 前30个差异表达基因的KEGG富集分析。(E) FOXO 信号通路。

Full size image

全尺寸图像

Pathological staging and survival analysis

病理分期与生存分析

The clinical importance of these genes named AKT and FOXO1 was analyzed using survival analysis and pathological staging based on gene expression levels in Fig.

图中基于基因表达水平，使用生存分析和病理分期分析了名为AKT和FOXO1的这些基因的临床重要性。

. The expressions of FOXO1 in 369 tumor tissues were significantly lower than those in 160 normal tissues in LIHC (Liver hepatocellular carcinoma), while the expression of AKT was no significant difference (

在肝细胞癌（LIHC）中，369个肿瘤组织中FOXO1的表达显著低于160个正常组织，而AKT的表达则无显著差异（

≥ 0.05). The results of pathological staging revealed that the expressions of AKT and FOXO1 were negatively correlated with the pathological stage of the tumor from stage I to IV. However, the F-value was 2.58 and the corresponding P-value was 0.0536 of AKT by using one-way ANOVA analysis, indicating that there was no significant difference (.

≥ 0.05）。病理分期结果显示，从肿瘤的I期到IV期，AKT和FOXO1的表达与病理分期呈负相关。然而，通过单因素方差分析，AKT的F值为2.58，相应的P值为0.0536，表明无显著差异（。

≥ 0.05) in the expression of AKT in different stages of LIHC, which was similar to FOXO1. The overall survival of patients with LIHC with high expression of AKT was significantly shorter than that of patients with low expressions of this gene, indicating that the prognosis of patients with low AKT expression was significantly better than that of patients with high expression.

肝细胞癌不同阶段中AKT的表达差异无统计学意义（P≥0.05），与FOXO1相似。高表达AKT的肝细胞癌患者的总生存期明显短于低表达患者，这表明低表达AKT的患者预后明显优于高表达患者。

On the contrary, the overall survival of patients with high expression of FOXO1 in tumor tissues was longer than that of patients with low expression of this gene. Therefore, AKT and FOXO1 might be the potential biomarkers and therapeutic targets related to the progression of LIHC..

相反，肿瘤组织中FOXO1高表达的患者总体生存期比该基因低表达的患者长。因此，AKT和FOXO1可能是与LIHC进展相关的潜在生物标志物和治疗靶点。

Fig. 6

图6

The expressions of AKT (A) and FOXO1 (B) in LIHC using box plots, violin plots and survival analysis.

使用箱线图、小提琴图和生存分析展示LIHC中AKT（A）和FOXO1（B）的表达。

Full size image

全尺寸图像

Molecular docking analysis

分子对接分析

To explore the specific binding mode of curcumin and its derivatives with AKT and FOXO1 proteins, molecular docking was conducted in this study by the Glide module in Schrodinger 2017. The crystal structure of AKT (PDB ID: 3O96, resolution: 2.70 Å, R

为了探索姜黄素及其衍生物与AKT和FOXO1蛋白的具体结合模式，本研究通过Schrodinger 2017中的Glide模块进行了分子对接。AKT的晶体结构（PDB ID：3O96，分辨率：2.70 Å，R值：...）。

free

免费

: 0.308) and FOXO1 (PDB ID: 6AEJ, resolution: 2.80 Å, R

：0.308）和FOXO1（PDB ID：6AEJ，分辨率：2.80 Å，R

free

自由的

: 0.222) were downloaded from the Protein Data Bank (

：0.222）从蛋白质数据库（Protein Data Bank）下载。

www.rcsb.org

). The conjugates

）。共轭物

H1-H6

, endogenous ligand, mono-carbonyl curcumin and curcumin were docked by standard precision (SP) glide and flexible dock according to the protocols. As shown in Table

，内源性配体、单羰基姜黄素和姜黄素按照标准精度（SP）滑移和柔性对接协议进行对接。如表中所示

, the docking score of

，对接得分

H1-H6

to AKT and FOXO1 proteins were stronger than endogenous ligand, mono-carbonyl curcumin and curcumin, which was conducive to playing a pivotal role in protein inhibition and stability. Importantly, the docking score of

对AKT和FOXO1蛋白的结合强于内源性配体、单羰基姜黄素和姜黄素，这有助于在蛋白抑制和稳定性方面发挥关键作用。重要的是，对接得分

was the highest among all of the conjugates, which was consistent with the result of the MTT assay. Conjugates

是所有结合物中最高的，这与MTT测定的结果一致。结合物

一级标题

was well extended into the active pocket and bound to key residues GLU85 (2.0 Å) and SER205 (2.3 Å) of AKT through intermolecular hydrogen bonds (No shown). As shown in Fig.

充分延伸到活性口袋中，并通过分子间氢键与AKT的关键残基GLU85（2.0 Å）和SER205（2.3 Å）结合（未显示）。如图所示。

, the NH

，NH

group of

一组

formed H-bonds with key residues HIS232, GLU234, TYR295, ARG316 and π-π stacking with key residue HIS231. These results of molecular docking may contribute to the difference in their anti-HCC activity.

与关键残基HIS232、GLU234、TYR295、ARG316形成氢键，并与关键残基HIS231发生π-π堆积。这些分子对接结果可能解释了它们抗肝细胞癌活性的差异。

Table 1 Docking results (Kcal/mol) of SP models of

表1 SP模型的对接结果（千卡/摩尔）

H1-H6

with AKT protein (3O96) and FOXO1 protein(6AEJ).

与AKT蛋白（3O96）和FOXO1蛋白（6AEJ）。

Full size table

全尺寸表格

Fig. 7

图7

The interaction pattern between derivative

衍生品之间的互动模式

一级标题

(A), curcumin (B), mono-carbonyl curcumin (C), endogenous ligand (D) and FOXO1 protein showed the electrostatic surface in the 2D and 3D representation from molecular docking.

(A)、姜黄素 (B)、单羰基姜黄素 (C)、内源性配体 (D) 和 FOXO1 蛋白在分子对接中展示了二维和三维表示中的静电表面。

一级标题

(green) and key residues (red) were represented in sticks and coloured by atom type.

(绿色) 和关键残基 (红色) 以棍状表示，并按原子类型着色。

Full size image

全尺寸图像

AKT/FOXO1 pathway

AKT/FOXO1 通路

Previous studies revealed that curcumin and mono-carbonyl curcumin esters suppressed HCC cancer development through inhibition of the expression of AKT protein

以往的研究表明，姜黄素和单羰基姜黄素酯通过抑制AKT蛋白的表达来抑制肝细胞癌的发展。

，

. To further assess whether derivative

. 为了进一步评估衍生品是否

had a similar blocking AKT function and how to regulate downstream signaling molecules, the cells were treated with different concentrations (0, 2, 4, 8 µM) of

具有类似的阻断AKT功能，并调节下游信号分子的方式，细胞接受不同浓度（0、2、4、8 µM）的处理。

一级标题

for 48 h, and cell lysates were prepared and analyzed by western blot. As shown in Fig.

持续48小时，并制备细胞裂解物并通过蛋白质印迹法进行分析。如图所示。

, H1 markedly suppressed the phosphorylation of AKT and up-regulated the expression of FOXO1 and P27 in a dose-dependent manner in HepG2 cells. In addition, compound

H1显著抑制了AKT的磷酸化，并以剂量依赖的方式上调了HepG2细胞中FOXO1和P27的表达。此外，化合物

一级标题

(4 µM) caused a significant increase in caspase-3 activation and inhibition in Bcl2 phosphorylation by western blotting with β-actin as the reference. This result indicated amino acid conjugates of mono-carbonyl curcumin

(4 µM) 通过 western blotting 检测，以 β-肌动蛋白为参照，显著增加了 caspase-3 的激活并抑制了 Bcl2 的磷酸化。这一结果表明单羰基姜黄素的氨基酸结合物

could inhibit cell proliferation and induce apoptosis through the AKT/FOXO1 pathway in hepatocellular carcinoma cells.

能够通过AKT/FOXO1通路抑制肝癌细胞的增殖并诱导凋亡。

Fig. 8

图8

Effect of derivative

衍生品效应

一级标题

(0, 2, 4, 8 µM) on p-AKT, FOXO1, p27, p-Bcl2, c-Caspase3 proteins at the cell level via western blot with β-actin as the reference. Data was represented by the mean ± SD of the three independent experiments. ***

(0, 2, 4, 8 µM)通过蛋白质印迹检测细胞水平上p-AKT、FOXO1、p27、p-Bcl2、c-Caspase3蛋白的变化，以β-肌动蛋白为参照。数据表示为三次独立实验的平均值±标准差。***

< 0.001, **

< 0.01, *

< 0.05, compared with the 0 µM group.

< 0.05，与 0 µM 组相比。

Full size image

全尺寸图像

Conclusion

结论

To look for a lead compound with potent anti-HCC activity, we evaluated the biological activity of novel amino acid conjugates of mono-carbonyl curcumin, known as

为了寻找一种具有强效抗肝细胞癌活性的先导化合物，我们评估了新型单羰基姜黄素氨基酸缀合物的生物活性，这些化合物被称为

H1-H6

. Remarkably,

。值得注意的是，

一级标题

exhibited more significant activity than that of curcumin and was equivalent to the positive drug sorafenib via MTT assay in HepG2 cells. A series of pharmacological assays containing cell proliferation by clone formation assay, cell migration by wound healing and transwell assay, cell apoptosis by JC-1 staining, and the underlying mechanism were further explored by transcriptome sequence, bioinformatics analysis, molecular docking, and western blotting.

在HepG2细胞中通过MTT实验显示出比姜黄素更显著的活性，并与阳性药物索拉非尼相当。通过克隆形成实验检测细胞增殖、伤口愈合和Transwell实验检测细胞迁移、JC-1染色检测细胞凋亡等一系列药理学实验，进一步通过转录组测序、生物信息学分析、分子对接和蛋白质印迹技术探讨了其潜在机制。

The results showed that .

结果显示。

一级标题

exerted greater potential than curcumin against liver cancer in vitro studies. The HepG2 cell xenograft model and H&E staining results also indicated that curcumin derivative

在体外研究中，对肝癌表现出比姜黄素更大的潜力。HepG2细胞异种移植模型和H&E染色结果也表明，姜黄素衍生物

had a better therapeutic effect on tumor growth. The antitumor mechanism may be related to the regulation of the AKT/FOXO1 pathway. All in all,

对肿瘤生长有较好的治疗效果，其抗肿瘤机制可能与调控AKT/FOXO1信号通路有关。总之，

was discovered and identified as a potent anti-hepatoma agent.

被发现并鉴定为一种有效的抗肝癌药物。

Data availability

数据可用性

Data will be made available on request. The data that support the findings of this study are available from the corresponding author Pan Yu upon reasonable request.

数据可根据要求提供。支持本研究发现的数据可由通讯作者潘宇在合理要求下提供。

References

参考文献

Agrawal, D. K. & Mishra, P. K. Curcumin and its analogues: potential anticancer agents.

Agrawal, D. K. & Mishra, P. K. 姜黄素及其类似物：潜在的抗癌剂。

Med. Res. Rev.

医学研究评论

(5), 818–860 (2010).

(5), 818–860（2010）。

Article

文章

PubMed

MATH

数学

Google Scholar

谷歌学术索

Noorafshan, A. & Ashkani-Esfahani, S. A review of therapeutic effects of Curcumin.

诺拉法珊，A. & 阿什卡尼-埃斯法哈尼，S. 姜黄素治疗效果的综述。

Curr. Pharm. Des.

当前药物设计

(11), 2032–2046 (2013).

PubMed

MATH

数学

Google Scholar

谷歌学术

Slika, L. & Patra, D. Traditional uses, therapeutic effects and recent advances of Curcumin: A Mini-Review.

图片，L. & Patra, D. 姜黄素的传统用途、治疗效果及最新进展：一篇小型综述。

Mini Rev. Med. Chem.

药物化学微型评论

(12), 1072–1082 (2020).

Article

文章

PubMed

MATH

数学

Google Scholar

谷歌学术

Zhao, S. et al. Recent advances of analogues of Curcumin for treatment of cancer.

赵, S. 等。姜黄素类似物在癌症治疗中的最新进展。

Eur. J. Med. Chem.

欧洲医药化学杂志

180

, 524–535 (2019).

，524-535页（2019年）。

Article

文章

ADS

PubMed

MATH

数学

Google Scholar

谷歌学术索

Anand, P. et al. Bioavailability of Curcumin: problems and promises.

Anand, P. 等。姜黄素的生物利用度：问题与前景。

Mol. Pharm.

分子药剂学

(6), 807–818 (2007).

Article

文章

PubMed

MATH

数学

Google Scholar

谷歌学术

Ireson, C. R. et al. Metabolism of the cancer chemopreventive agent Curcumin in human and rat intestine.

Ireson, C. R. 等。抗癌化学预防剂姜黄素在人和大鼠肠道中的代谢。

Cancer Epidemiol. Biomarkers Prev.

癌症流行病学、生物标志物与预防。

(1), 105–111 (2002).

PubMed

MATH

数学

Google Scholar

谷歌学术索

Zhao, C., Liu, Z. & Liang, G. Promising Curcumin-based drug design: mono-carbonyl analogues of Curcumin (MACs).

赵, C., 刘, Z. & 梁, G. 有前景的姜黄素类药物设计：姜黄素的单羰基类似物 (MACs)。

Curr. Pharm. Des.

当前药物设计

(11), 2114–2135 (2013).

PubMed

MATH

数学

Google Scholar

谷歌学术

Pan, Z. et al. Synthesis and cytotoxic evaluation of monocarbonyl analogs of Curcumin as potential Anti-Tumor agents.

潘，Z. 等。单羰基姜黄素类似物的合成及其作为潜在抗肿瘤剂的细胞毒评价。

Drug Dev. Res.

药物开发研究。

(1), 43–49 (2016).

Article

文章

PubMed

PubMed Central

MATH

数学

Google Scholar

谷歌学术

Reddy, A. R. et al. A comprehensive review on SAR of Curcumin.

Reddy, A. R. 等。姜黄素的SAR全面综述。

Mini Rev. Med. Chem.

药物化学微型评论

(12), 1769–1777 (2013).

Article

文章

PubMed

MATH

数学

Google Scholar

谷歌学术

Rodrigues, F. C., Anil Kumar, N. V. & Thakur, G. Developments in the anticancer activity of structurally modified Curcumin: an up-to-date review.

罗德里格斯，F. C.，阿尼尔·库马尔，N. V.，塔库尔，G. 结构修饰的姜黄素抗癌活性的研究进展：最新综述。

Eur. J. Med. Chem.

欧洲医学化学杂志

177

, 76–104 (2019).

，76-104页（2019年）。

Article

文章

PubMed

MATH

数学

Google Scholar

谷歌学术

Yu, P. et al. Design, synthesis, and antitumor evaluation of novel Mono-Carbonyl Curcumin analogs in hepatocellular carcinoma cell. Pharmaceuticals (Basel), 15(8) (2022).

余鹏等。新型单羰基姜黄素类似物在肝细胞癌细胞中的设计、合成及抗肿瘤评估。《制药》（巴塞尔），2022年，第15卷第8期。

Cao, W. et al. Discovery of novel Mono-Carbonyl Curcumin derivatives as potential Anti-Hepatoma agents.

曹，W. 等。新型单羰基姜黄素衍生物作为潜在抗肝癌药物的发现。

Molecules

分子

，

(19). (2023).

Kelly, B. & Pearce, E. L. Amino assets: how amino acids support immunity.

凯利，B. & 皮尔斯，E. L. 氨基资产：氨基酸如何支持免疫。

Cell. Metab.

细胞代谢

(2), 154–175 (2020).

Article

文章

PubMed

Google Scholar

谷歌学术

Durante, W. Amino acids in circulatory function and health.

During, W. 氨基酸在循环功能和健康中的作用。

Adv. Exp. Med. Biol.

高级实验医学与生物学

1265

一千二百六十五

, 39–56 (2020).

，39-56页（2020年）。

Article

文章

PubMed

MATH

数学

Google Scholar

谷歌学术

Lau, J. L. & Dunn, M. K. Therapeutic peptides: historical perspectives, current development trends, and future directions.

刘，J. L. & 邓，M. K. 治疗性肽：历史视角、当前发展趋势与未来方向。

Bioorg. Med. Chem.

生物有机化学与药物化学

(10), 2700–2707 (2018).

Article

文章

PubMed

MATH

数学

Google Scholar

谷歌学术索

Fu, X. Y. et al. Three rounds of Stability-Guided optimization and systematical evaluation of oncolytic peptide LTX-315.

傅晓燕等。三轮稳定性引导优化及溶瘤肽LTX-315的系统评估。

J. Med. Chem.

药物化学杂志

(5), 3885–3908 (2024).

Article

文章

PubMed

MATH

数学

Google Scholar

谷歌学术

Yin, H. et al. The hybrid oncolytic peptide NTP-385 potently inhibits adherent cancer cells by targeting the nucleus.

阴，H. 等。杂交溶瘤肽 NTP-385 通过靶向细胞核有效抑制贴壁癌细胞。

Acta Pharmacol. Sin

中国药理学报

。

(1), 201–210 (2023).

Article

文章

PubMed

MATH

数学

Google Scholar

谷歌学术

Yin, H. et al. Design, synthesis and anticancer evaluation of novel oncolytic peptide-chlorambucil conjugates.

阴，H. 等。新型溶瘤肽-氯丙脒结合物的设计、合成及抗癌评估。

Bioorg. Chem.

生物有机化学

138

, 106674 (2023).

，106674（2023）。

Article

文章

PubMed

MATH

数学

Google Scholar

谷歌学术索

Arifian, H. et al. Amino-Acid-Conjugated natural compounds: aims, designs and results.

阿里菲安，H. 等。氨基酸结合的天然化合物：目标、设计与结果。

Molecules

分子

，

(21). (2022).

Manjunatha, J. R. et al. Synthesis of amino acid conjugates of Tetrahydrocurcumin and evaluation of their antibacterial and anti-mutagenic properties.

Manjunatha, J. R. 等。四氢姜黄素氨基酸缀合物的合成及其抗菌和抗诱变特性的评估。

Food Chem.

食品化学

139

一百三十九

(1–4), 332–338 (2013).

Article

文章

PubMed

Google Scholar

谷歌学术索

Ni, J. et al. Preparation of valine-curcumin conjugate and its in vitro antibacterial and antitumor activity and in vivo biological effects on American Eels (Anguilla rostrata).

倪, J. 等。缬氨酸-姜黄素结合物的制备及其体外抗菌、抗肿瘤活性和对美洲鳗鲡（Anguilla rostrata）的体内生物学效应。

Fish. Shellfish Immunol.

鱼类和贝类免疫学

149

, 109615 (2024).

，109615（2024）。

Article

文章

PubMed

MATH

数学

Google Scholar

谷歌学术

Kim, M. K. et al. In vitro solubility, stability and permeability of novel quercetin-amino acid conjugates.

金，M. K. 等。新型槲皮素-氨基酸结合物的体外溶解性、稳定性和渗透性。

Bioorg. Med. Chem.

生物有机化学与药物化学

(3), 1164–1171 (2009).

Article

文章

PubMed

MATH

数学

Google Scholar

谷歌学术索

Fang, L. et al. Combination of amino acid/dipeptide with nitric oxide donating oleanolic acid derivatives as PepT1 targeting antitumor prodrugs.

方，L. 等。氨基酸/二肽与一氧化氮供体齐墩果酸衍生物组合作为PepT1靶向抗肿瘤前药。

J. Med. Chem.

药物化学杂志

(3), 1116–1120 (2014).

Article

文章

MathSciNet

数学评论

PubMed

MATH

数学

Google Scholar

谷歌学术索

Dubey, S. K. et al. Design, synthesis and characterization of some bioactive conjugates of Curcumin with Glycine, glutamic acid, valine and demethylenated piperic acid and study of their antimicrobial and antiproliferative properties.

Dubey, S. K. 等。姜黄素与甘氨酸、谷氨酸、缬氨酸和去亚甲基胡椒酸的一些生物活性结合物的设计、合成与表征及其抗菌和抗增殖特性的研究。

Eur. J. Med. Chem.

欧洲医学化学杂志

四十三

(9), 1837–1846 (2008).

Article

文章

PubMed

Google Scholar

谷歌学术索

Qi, Y. K., Zheng, J. S. & Liu, L. Mirror-image protein and peptide drug discovery through mirror-image phage display.

齐，Y. K.，郑，J. S.，刘，L. 通过镜像噬菌体展示进行镜像蛋白质和肽类药物发现。

Chem

化学

(8), 2390–2407 (2024).

Article

文章

MATH

数学

Google Scholar

谷歌学术

Yu, P. et al. Synthesis and antitumor activity of C-3(R) hydroxy modified betulinic acid derivatives.

余鹏等。C-3(R) 羟基修饰的白桦酸衍生物的合成及抗肿瘤活性。

Chem. Nat. Compd.

化学与自然化合物

(6), 1080–1084 (2019).

Article

文章

MATH

数学

Google Scholar

谷歌学术

Cao, W. et al. Autophagy up-regulated by MEK/ERK promotes the repair of DNA damage caused by aflatoxin B1.

曹，W. 等。MEK/ERK上调的自噬促进黄曲霉毒素B1引起的DNA损伤修复。

Toxicol. Mech. Methods

毒理学机制与方法

。

(2), 87–96 (2022).

Article

文章

PubMed

MATH

数学

Google Scholar

谷歌学术索

Cao, W. et al. Curcumin reverses hepatic epithelial mesenchymal transition induced by trichloroethylene by inhibiting IL-6R/STAT3.

曹，W. 等。姜黄素通过抑制IL-6R/STAT3逆转三氯乙烯诱导的肝上皮间质转化。

Toxicol. Mech. Methods

毒理学机制与方法

。

(8), 589–599 (2021).

Article

文章

PubMed

Google Scholar

谷歌学术

Yu, P. et al.

余，P. 等

Design, synthesis and antitumor evaluation of novel Quinazoline analogs in hepatocellular carcinoma cell

新型喹唑啉类化合物在肝细胞癌细胞中的设计、合成及抗肿瘤评估

。

J. Mol. Struct.

分子结构杂志

, 1268. (2022).

，1268.（2022）。

Wang, P. F. et al. Identification of novel B-Raf(V600E) inhibitors employing FBDD strategy.

王，P. F. 等。利用FBDD策略鉴定新型B-Raf(V600E)抑制剂。

Biochem. Pharmacol.

生化药理学

132

一百三十二

, 63–76 (2017).

，63-76页（2017年）。

Article

文章

ADS

PubMed

MATH

数学

Google Scholar

谷歌学术

Kong, Y. et al. Discovery and structural optimization of 9-O-phenylsulfonyl-berberines as new lipid-lowering agents.

孔烨等。发现并结构优化9-O-苯磺酰基小檗碱作为新型降脂剂。

Bioorg. Chem.

生物有机化学

121

, 105665 (2022).

，105665（2022）。

Article

文章

ADS

PubMed

MATH

数学

Google Scholar

谷歌学术

Sherman, B. T. et al. DAVID: a web server for functional enrichment analysis and functional annotation of gene lists (2021 update).

Sherman, B. T. 等。DAVID：用于基因列表功能富集分析和功能注释的网络服务器（2021年更新）。

Nucleic Acids Res.

核酸研究。

(W1), W216–W221 (2022).

Article

文章

ADS

PubMed

PubMed Central

MATH

数学

Google Scholar

谷歌学术

Tang, Z. et al. GEPIA2: an enhanced web server for large-scale expression profiling and interactive analysis.

唐，Z. 等。GEPIA2：一个增强的网页服务器，用于大规模表达谱分析和交互式分析。

Nucleic Acids Res.

核酸研究

(W1), W556–W560 (2019).

Article

文章

PubMed

PubMed Central

Google Scholar

谷歌学术

Yu, P. et al. Identification of dihydroorotate dehydrogenase as a protein target of ginkgolic acid by molecular Docking and dynamics.

余鹏等。通过分子对接和动力学鉴定二氢乳清酸脱氢酶为银杏酸的蛋白质靶标。

J. Mol. Struct.

分子结构杂志

, 1220. (2020).

，1220.（2020）。

Wu, W. I. et al. Crystal structure of human AKT1 with an allosteric inhibitor reveals a new mode of kinase Inhibition.

吴伟等。人源AKT1与变构抑制剂的晶体结构揭示了一种新的激酶抑制模式。

PLoS One

。

(9), e12913 (2010).

Article

文章

ADS

PubMed

PubMed Central

MATH

数学

Google Scholar

谷歌学术

Yu, P., Cao, W. & Wang, Y. Dynamics simulation and in vitro studies of betulinic acid derivative with liver X receptor.

余鹏、曹伟和王莹。熊果酸衍生物与肝X受体的动力学模拟及体外研究。

J. Biomol. Struct. Dyn.

生物分子结构与动力学杂志

, pp. 1–10. (2023).

，第1-10页。（2023年）

Mou, S. et al. Curcumin inhibits cell proliferation and promotes apoptosis of laryngeal cancer cells through Bcl-2 and PI3K/Akt, and by upregulating miR-15a.

牟等人。姜黄素通过Bcl-2和PI3K/Akt途径抑制喉癌细胞增殖并促进其凋亡，并通过上调miR-15a。

Oncol. Lett.

肿瘤学快报

(4), 4937–4942 (2017).

Article

文章

PubMed

PubMed Central

MATH

数学

Google Scholar

谷歌学术

Rana, C. et al. Downregulation of PI3-K/Akt/PTEN pathway and activation of mitochondrial intrinsic apoptosis by diclofenac and Curcumin in colon cancer.

Rana, C. 等。双氯芬酸和姜黄素对结肠癌中PI3-K/Akt/PTEN通路的下调及线粒体内在凋亡的激活。

Mol. Cell. Biochem.

分子细胞生物化学

402

(1–2), 225–241 (2015).

Article

文章

MathSciNet

数学评论

PubMed

MATH

数学

Google Scholar

谷歌学术

Download references

下载参考文献

Acknowledgements

致谢

This work was supported by the Scientific Research Foundation for High-level Talents of Anhui University of Science and Technology (NO. 2022yjrc69), Joint Research Center of Occupational Medicine and Health, Institute of Grand Health, Hefei Comprehensive National Science Center (Anhui University of Science and Technology) (NO.

这项工作得到了安徽理工大学高层次人才科学研究基金（NO. 2022yjrc69）、合肥综合性国家科学中心大健康研究院职业医学与健康联合研究中心（安徽理工大学）（NO.

OMH-2023-14), Medical Special Cultivation Project of Anhui University of Science and Technology (NO. YZ2023H1B005), Natural Science Research Project of Anhui Educational Committee (No. KJ2021A0436), Anhui Provincial Natural Science Foundation (NO. 2108085QH381)..

OMH-2023-14），安徽理工大学医学专项培育项目（编号：YZ2023H1B005），安徽省教育厅自然科学研究项目（编号：KJ2021A0436），安徽省自然科学基金（编号：2108085QH381）。

Author information

作者信息

Authors and Affiliations

作者与所属机构

College of Public Health, Anhui University of Science and Technology, Hefei, 230000, China

安徽理工大学公共卫生学院，合肥，230000，中国

Weiya Cao, Pan Yu, Yongchang Cao, Shuhui Feng & Nan Yin

曹伟亚、余攀、曹永昌、冯淑惠、尹楠

Joint Research Center of Occupational Medicine and Health, Institute of Grand Health, Hefei Comprehensive National Science Center, Hefei, 230000, China

中国合肥，合肥综合性国家科学中心，大健康研究院，职业医学与健康联合研究中心，邮编230000

Weiya Cao

曹伟亚

The First Affiliated Hospital of Anhui University of Science and Technology (Huainan First People’S Hospital), Huainan, 232001, China

安徽理工大学第一附属医院（淮南市第一人民医院），淮南，232001，中国

Pan Yu

盘屿

Authors

作者

Weiya Cao

曹伟亚

View author publications

查看作者的出版物

You can also search for this author in

您还可以搜索该作者在

PubMed

Google Scholar

谷歌学术

Pan Yu

潘宇

View author publications

查看作者的出版物

You can also search for this author in

您还可以搜索此作者在

PubMed

Google Scholar

谷歌学术索

Yongchang Cao

永昌曹

View author publications

查看作者的出版物

You can also search for this author in

您还可以搜索此作者在

PubMed

Google Scholar

谷歌学术索

Shuhui Feng

冯淑慧

View author publications

查看作者的出版物

You can also search for this author in

您还可以搜索此作者在

PubMed

Google Scholar

谷歌学术

Nan Yin

南音

View author publications

查看作者的出版物

You can also search for this author in

您还可以搜索此作者在

PubMed

Google Scholar

谷歌学术搜索

Contributions

贡献

W.C. and P.Y. conceived and designed the experiments. W.C., Y.C., S.F., N.Y. performed the experiments. W.C. and P.Y. analyzed the data and wrote the paper. All authors reviewed and approved the manuscript .

W.C. 和 P.Y. 构思并设计了实验。W.C.、Y.C.、S.F.、N.Y. 进行了实验。W.C. 和 P.Y. 分析了数据并撰写了论文。所有作者审阅并批准了手稿。

Corresponding author

通讯作者

Correspondence to

致信给

Pan Yu

盘屿

。

Ethics declarations

伦理声明

Competing interests

竞争利益

The authors declare no competing interests.

作者声明不存在竞争性利益。

Additional information

附加信息

Publisher’s note

出版商说明

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Springer Nature 对已发布地图中的管辖权声明和机构隶属关系保持中立态度。

Electronic supplementary material

电子补充材料

Below is the link to the electronic supplementary material.

以下是电子补充材料的链接。

Supplementary Material 1

补充材料1

Supplementary Material 2

补充材料 2

Rights and permissions

权利与许可

Open Access

开放获取

This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material.

本文根据知识共享署名-非商业性使用-禁止演绎 4.0 国际许可协议获得许可，该协议允许您在任何媒介或格式中进行非商业性的使用、分享、分发和复制，只要您对原作者和来源给予适当的署名，提供指向知识共享许可协议的链接，并说明是否对授权材料进行了修改。

You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.

根据本许可，您无权分享从本文或其部分内容衍生的改编材料。本文中的图像或其他第三方材料包含在文章的Creative Commons许可中，除非在材料的署名行中另有说明。如果材料未包含在文章的Creative Commons许可中，并且您的预期用途不被法律规定允许或超出了允许的使用范围，您需要直接从版权持有人处获得许可。

To view a copy of this licence, visit .

要查看此许可证的副本，请访问。

http://creativecommons.org/licenses/by-nc-nd/4.0/

。

Reprints and permissions

重印和权限

About this article

关于本文

Cite this article

引用本文

Cao, W., Yu, P., Cao, Y.

曹，W.，余，P.，曹，Y.