Wang et al. BMC Complementary Medicine and Therapies (2020) 20:29 BMC Complementary https://doi.org/10.1186/s12906-020-2831-y Medicine and Therapies RESEARCH ARTICLE Open Access Synthesis of crocetin derivatives and their potent inhibition in multiple tumor cells proliferation and inflammatory property of macrophage Mao-Ze Wang1†, Jin Gao2,3,4†, Yang Chu1, Jie Niu1, Ming Chen2, Qiang Shang3,4, Li-Hua Peng1,2* and Zhi-Hong Jiang2* Abstract Background: Crocetin is a major active component of saffron, which has a wide range of pharmacological effects. However, due to its low solubility, the pharmacological effects of crocetin cannot be better utilized. Methods: In this study, we modified the chemical structure of crocetin by conjugating with ethylamine and 4- Fluorbenzylamine to enhance its solubility and biological activities. The solubility and the influence of synthesized derivatives on the proliferation of tumor cells and the inflammatory effect of macrophage were investigated. Results: It was shown that, compared with the crocetin, the synthesized derivatives have much higher solubility. Moreover, the inhibitory effect of the derivatives on varieties of tumor cells, including human ovarian carcinoma cell line, human lung cancer cell line, rat melanoma cell line was enhanced after the modification. Besides that, the derivatives were improved for the anti-inflammatory efficacy with the cytotoxicity decreased. Conclusions: The synthesized derivatives were shown for their good solubility and the great potential in tumor and inflammation treatment. Keywords: Solubility, Crocetin derivatives, Anti-tumor effect, Inflammation inhibition Background one of the main effective components [2], with polyunsat- Saffron, a spice and a food colorant derived from the urated conjugate oleic acid structure. Many studies have flower of Crocus sativus, is widely cultivated in Iran, shown that crocetin has widely pharmacological effects, Greece, Italy, Spain, India, China, Japan and other coun- which contains anti-oxidation [4], anti-cancer [5], hepato- triesasanherb[1]. After collection and dehydration, it protection [6, 7] However, consisting of a C-20 carbon can be used mainly as the medicinal parts [2]. The history chain with seven double bonds and a carboxylic acid of the Saffron application in clinic can be traced back to group at each terminal parts of the molecule, crocetin is about 2300 Before Christ [3]. Saffron contains a variety of insoluble in water. Solving this problem is the key to pro- ingredients, including carotenoids, glycosides, flavonoids, mote the medicinal application of this natural product. amino acids, trace elements, etc. Among them, crocetin is Currently, it has been reported that the use of biomaterials such as poly (lactic-co-glycolic acid) and dendrimers can improve the bioavailability of the crocetin. However, the * Correspondence: [email protected]; [email protected] †Mao-Ze Wang and Jin Gao contributed equally to this work. stability and production rates are still to be improved for 1Institute of Pharmaceutics, College of Pharmaceutical Sciences, Zhejiang these biomaterials and nanotechnology based strategies [8, University, 866# Yuhangtang Road, Hangzhou 310058, People’s Republic of 9]. In our previous study [10], it has been demonstrated China 2State Key Laboratory of Quality Research in Chinese Medicine, Macau that the conjugation of organic compounds can help de- University of Science and Technology, Avenida Wai Long Taipa, Macau, rivatives to form stable hydrogen bond by lowing solvation People’s Republic of China energy [10, 11], which in turn increases the solubility of Full list of author information is available at the end of the article © The Author(s). 2020 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Wang et al. BMC Complementary Medicine and Therapies (2020) 20:29 Page 2 of 8 the compounds. In present study, by conjugating the cro- Synthesis of crocetin derivatives cetin with ethylamine and 4-Fluorobenzylamine, the solu- The synthesis route of crocetin derivatives was showed in bility and bioactivity of crocetin were expected to be Fig. 1. Briefly, HOBt (2 equiv) were added to EDCI (1.5 improved. We evaluated the solubility, anti-proliferation equiv) at 0 °C, and Et3N (1.5 equiv) and 4- activity and anti-inflammatory property of the synthesized fluorobenzylamine (1 equiv) were added subsequently. The derivatives. Results of this strategy is expected to provide mixture solution was proceed at 0 °C for about 6 h and then novel evidence for the property improvement and medi- at 25 °C for 12 h. After that, the mixture solution was di- cinal application of crocetin. luted with dichloromethane. And then the unreacted or- ganic compound in mixture was washed with 2.5% HCl and 7.5% NaHCO3 (15 mL, three times). The product of synthe- Methods sized crocetin derivatives were obtained by evaporated Materials under reduced pressure. Then, crocetin derivatives were 1-[3-(dimethylamino)-propyl]-3-ethylcarbodiimide hydro- purified by column chromatography. chloride (EDCI); petroleum ether (PE); 4-fluorobenzylamine, ethylamine; 1-hydroxybenzotrizole (HOBt); Dulbecco’s Solubility assays modified Eagle’s medium (DMEM); Et3N, trimethylamine; To determine the solubility of crocetin and the synthe- the fetal bovine serum (FBS) and trypsin were purchased sized derivatives in methanol, we established the stand- from the Gibco BRL (Gaithersberg,MD).BCAproteinassay ard curve by measuring its maximum absorbance with kit and Cell Counting Kit-8 were provided by Sangon Bio- ultraviolet spectrophotometer. First, dissolving the sam- tech (Shanghai) Co., Ltd. Nitric Oxide assay kit were pur- ples in water. And then the solution was ultrasounded chased from Beyotime Institute of Biotechnology, (Jiangsu, until the supersaturated solution was generated. After China). The mouse macrophage cell line and the cancer cell that, supersaturated solution was centrifugated at 12,000 lines were obtained from the Shanghai Institute for rpm for 15 min. The supernatant of the solution was col- Biological Sciences, CAS (Shanghai). Other chemicals and lected and freeze-dried, followed by using methanol to reagents were of analytical grade. re-dissolve for UV detection. The concentration of the Fig. 1 Synthesis and chemical structures of compounds A and B Wang et al. BMC Complementary Medicine and Therapies (2020) 20:29 Page 3 of 8 solution could be figured out by the standard curve. Result Upon that, we could obtain the solubility of the crocetin Solubility of the crocetin derivatives and its synthesized derivatives. The solubility of the synthesized derivatives was shown in Fig. 1. The results showed that the solubility in water Cell culture of crocetin was 1.238 μg/mL, while the solubility in The mouse macrophage cell line and the cancer cell water of compound A and B were 19.55, 12.28 times lines were cultured in the DMEM medium containing more than that of crocetin, respectively (Table 1). The 10% Fetal Bovine Serum (FBS) at 37 °C, in humidified at- significant increase in the solubility of these synthesized mosphere containing 5% CO2. The cell medium was re- derivatives indicated that the chemical modification of placed every two days. Once cells reached 70–80% these groups, which enhanced hydrophilicity and in- confluence, they could be used in the next experiments. creased hydrogen bond formation [12], is an effective strategy to increase solubility of crocetin. Anti-proliferation assay The anti-proliferation effects of compounds on tumor cells were calculated by CCK-8 assay using Cell Count- Anti-tumor effects of the crocetin derivatives ing Kit-8 (Sangon Biotech, Shanghai) according to man- The influence of these compounds in the viability of three ufacturer’s protocol. Cancer cell lines (SKOV3, A549 kinds of cancer cell lines was investigated. With different and B16F10 cells) were seeded in 96-well plates. 5 × 103 solubility, the highest concentration of the tested com- cancer cells were added to each well and incubated over- pounds in the mixture of water with 1% DMSO was tested night for proliferation. And then in test groups, they as 15.24 μg/mL, 60.98 μg/ml and 60.98 μg/mL. As shown were treated with different concentrations of synthesized in Fig. 2, we first investigated the effects of different con- derivatives, which are dissolved in DMSO and diluted by centrations of crocetin and synthesized derivatives on the cell medium. The group, whose cells were incubated proliferation of A549, a lung cancer cell [13]. After one without compounds, was regard as control group. After day of incubation of the compound with the cells, it was incubated for 24 h or 48 h, the cell medium was changed found that the cells viability was significantly de- by fresh medium (90 μl) and 10 μl of CCK-8 assay re- creased in contrast to that of solvent treated group. agent. The cells were incubated at 37 °C for 1 h. The OD The crocetin expressed the highest suppression per- values of each well were measured at 450 nm. centage at the 15.24 μg/mL, and the cells viability was decreased to 56%. While the maximum inhibitory effi- Evaluation of the anti-inflammatory effect in LPS- ciency of the compound A and B was expressed as stimulated macrophages 48.52 and 38.41%, at the 15.24 and 60.98 μg/mL, The anti-inflammatory effect was evaluated by using Ni- respectively. tric Oxide Assay kit. The RAW 264.7 cells with cell con- Then we investigated the influence of the compound centration 4 × 105 cells/ml were seeded in a 48-well on B16F10 cell proliferation.