This paper presents the successful application of ultrasound-assisted packed-bed (UAE-PB) method for the extraction of hypericin from the Hypericum perfuratum L. The Soxhlet system was utilized for the determination of suitable solvent from ethanol, methanol or from the mixture of different proportions of ethanol-methanol. The mixture of 50:50 v/v ethanol-methanol was obtained to be the most suitable solvent since it led to the highest extraction amount of hypericin. The extraction amount of hypericin increased by 13.6% and 21.4% when the solvent changed from pure methanol to the mixture of 50:50 v/v ethanol-methanol for the extraction time of 3 and 8 h, respectively. Subsequently, the extraction was conducted through the UAE-PB, and the effects of temperature, time, and the ratio of solvent to the dried plant were studied. The response surface method (RSM) was used to investigate the effect of parameters on the extraction in the UAE-PB system. At the temperature of 60 °C, extraction time of 105 min, and the solvent to plant ratio of 15.3, the maximum extraction yield of hypericin was achieved. In the optimal conditions, the amount of extraction was 0.112 mg hypericin/g dried plant, which was in accordance with the optimized predicted value (0.111 mg hypericin/g dried plant) from Design-Expert software.
Given that p53 is a tumor suppressor that plays a central role in the cellular response to DNA damage and that more than 50% of all cancers have mutated p53, the wider utility of photodynamic therapy (PDT) in the treatment of cancer will depend on an understanding of whether p53 status modulates response to PDT. In this study, we investigated the photosensitivity of isogenic cell lines that differ only in their p53 status to PDT using hypericin as the photosensitizer.
Photodynamic therapy (PDT) has emerged as a capable therapeutic modality for the treatment of cancer. PDT is a targeted cancer therapy that reportedly leads to tumor cell apoptosis and/or necrosis by facilitating the secretion of certain pro-inflammatory cytokines and expression of multiple apoptotic mediators in the tumor microenvironment. In addition, PDT also triggers oxidative stress that directs tumor cell killing and activation of inflammatory responses. However, the cellular and molecular mechanisms underlying the role of PDT in facilitating tumor cell apoptosis remain ambiguous. Here, we investigated the ability of PDT in association with hypericin (HY) to induce tumor cell apoptosis by facilitating the induction of reactive oxygen species (ROS) and secretion of Th1/Th2/Th17 cytokines in human hepatocellular liver carcinoma cell line (HepG2) cells. To discover if any apoptotic mediators were implicated in the enhancement of cell death of HY-PDT-treated tumor cells, selected gene profiling in response to HY-PDT treatment was implemented. Experimental results showed that interleukin (IL)-6 was significantly increased in all HY-PDT-treated cells, especially in 1 μg/ml HY-PDT, resulting in cell death. In addition, quantitative real-time PCR analysis revealed that the expression of apoptotic genes, such as BH3-interacting-domain death agonist (BID), cytochrome complex (CYT-C) and caspases (CASP3, 6, 7, 8 and 9) was remarkably higher in HY-PDT-treated HepG2 cells than the untreated HepG2 cells, entailing that tumor destruction of immune-mediated cell death occurs only in PDT-treated tumor cells. Hence, we showed that HY-PDT treatment induces apoptosis in HepG2 cells by facilitating cytotoxic ROS, and potentially recruits IL-6 and apoptosis mediators, providing additional hints for the existence of alternative mechanisms of anti-tumor immunity in hepatocellular carcinoma, which contribute to long-term suppression of tumor growth following PDT.