Chrysophanol

TSG, emodin, chrysophanol, physcion, rhein, and dimethylsulfoxide (DMSO) and water soluble β-estradiol were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA).

The preparation of Gemini-Chr NPs was carried out by nanoprecipitation method [22 (link)]. Briefly, 5 mg of chrysophanol (CAS no.: 481–74-3, Sigma Aldrich, USA) and 100 mg of methoxy-poly (ethylene glycol) urethane Gemini surfactant were dissolved in 5 mL methanol (Merck). After evaporation of methanol by rotatory evaporator, the solution is filtered by 0.2 μm syringe filter, lyophilized, and stored at 4 °C until further use. chrysophanol was dissolved in phosphate-buffered saline (PBS) before each treatment. Dynamic light scattering (DLS), which is applied for the physicochemical characterization of nanoparticles, was used to measure the zeta potential as well as the hydrodynamic diameter of the Gemini-Chr NPs. Additionally, scanning electron microscopy (SEM) achieved detailed visual image of the Gemini-Chr NPs with high-quality and spatial resolution for characterization and a better understanding of cytotoxicity mechanism [23 (link)]. The structural representation of a novel Gemini-Chr NPs is shown in Fig. 1.Fig. 1

The structural representation of a novel Gemini-Chr NPs

Anti-claudin 5 was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Horseradish peroxidase conjugated goat anti-rabbit and goat anti-mouse IgGs were purchased from Novus Biological (Centennial, CO, USA). Trizol was obtained from Invitrogen (Invitrogen, Carlsbad, CA, USA) and reverse transcriptional polymerase chain reaction (RT-PCR) kit was obtained from Promega (Promega, Madison WI, USA). Sennoside A, emodin, chrysophanol, aloe-emodin, rhein, glycyrrhizin acid, liquiritigenin, isoliquiritigenin, and other reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA). The reagents for ultra-performance liquid chromatography (UPLC) analysis were methanol (Junsei for the high-performance liquid chromatography (HPLC), acetonitrile (JT BAKER for the HPLC), and then Water (Tertiary distilled water).

Berberine hydrochloride was purchased from TCI Co., Ltd. (Tokyo, Japan). Chrysin was obtained from Acros Organics (Geel, Belgium). Chrysophanol was purchased from Sigma-Aldrich (St. Louis, MI, USA). Vascular endothelial growth factor (VEGF) was bought from B & D Systems (Minneapolis, MN, USA). Rhubarb (dried stem and root from Rheum palmatum LINN) was a product of Da Rong Co., Ltd. (Tao Yuan City, Taiwan) (batch number: DK1070932). Scutellaria root (dried root from Scutellaria baicalensis Georgi) was the product of He Kang Chinese Medicine Co., Ltd. (New Taipei City, Taiwan) (batch number: 0704). Phellodendron bark (dried bark from Phellodendron amurense Ruprecht) was the product of Jin Rong Co., Ltd. (New Taipei City, Taiwan) (batch number: AG80303). Coptidis rhizome (dried rhizome of Coptis chinensis Franch) was the product of Fu Ji Co., Ltd. (Kaohsiung City, Taiwan) (batch number: FG0013). Further identification analysis of these plant-based materials was done by HPLC, as described below. The raw herb materials were ground into fine powder using a coffee grinder (Model: ECG3003S, Electrolux, New Taipei City, Taiwan). The milled powder was further sieved using ultrafine 100 mesh stainless steel filter and stored in a dry cabinet. The recipes of two different types of “San Huang Powder” are listed in Table 1.