Conventional chemotherapeutic agents for colorectal cancer (CRC) cause systemic side effects and eventually become less efficacious owing to the development of drug resistance in cancer cells. Therefore, new therapeutic regimens have focused on the use of natural products. The anticancer activity of several parts ofCalotropis giganteahas been reported; however, the effects of its stem bark extract on inhibition of cancer cell proliferation have not yet been examined. In this study, the anticancer activity ofC.giganteastem bark extract, both alone and in combination with 5-fluorouracil (5-FU), was evaluated. A crude ethanolic extract was prepared from dry, powderedC.giganteabarks using 95% ethanol. This was then partitioned to obtain dichloromethane (CGDCM), ethyl acetate, and water fractions. Quantitative analysis of the constituent secondary metabolites and calotropin was performed. These fractions exhibited cytotoxicity in HCT116 and HT-29 cells, with CGDCM showing the highest potency in both the cell lines. A combination of CGDCM and 5-FU significantly enhanced the cytotoxic effect. Moreover, the resistance of normal fibroblast, HFF-1, cells to this combination demonstrated its safety in normal cells. The combination significantly enhanced apoptosis through the mitochondria-dependent pathway. Additionally, the combination reduced adenosine triphosphate production and increased the production of reactive oxygen species, demonstrating the mechanisms involved in the induction of apoptosis. Our results suggest that CGDCM is a promising anti-cancer agent and may enhance apoptosis induction by 5-FU in the treatment of CRC, while minimizing toxicity toward healthy cells.

Several fractions ofCalotropis giganteaextracts have been proposed to have potential anticancer activity in many cancer models. The present study evaluated the anticancer activity ofC. giganteastem bark extracts in liver cancer HepG2 cells and diethylnitrosamine (DEN)-induced primary liver cancer in rats. The carcinogenesis model induced by DEN administration has been widely used to study pathophysiological features and responses in rats that are comparable to those seen in cancer patients. The dichloromethane (CGDCM), ethyl acetate, and water fractions obtained from partitioning crude ethanolic extract were quantitatively analyzed for several groups of secondary metabolites and calactin contents. A combination ofC. giganteastem bark extracts with doxorubicin (DOX) was assessed in this study to demonstrate the enhanced cytotoxic effect to cancer compared to the single administration. The combination of DOX and CGDCM, which had the most potential cytotoxic effect in HepG2 cells when compared to the other three fractions, significantly increased cytotoxicity through the apoptotic effect with increased caspase-3 expression. This combination treatment also reduced ATP levels, implying a correlation between ATP and apoptosis induction. In a rat model of DEN-induced liver cancer, treatment with DOX,C. giganteaat low (CGDCM-L) and high (CGDCM-H) doses, and DOX + CGDCM-H for 4 weeks decreased the progression of liver cancer by lowering the liver weight/body weight ratio and the occurrence of liver hyperplastic nodules, fibrosis, and proliferative cells. The therapeutic applications lowered TNF-α, IL-6, TGF-β, and α-SMA inflammatory cytokines in a similar way, implying that CGDCM had a curative effect against the inflammation-induced liver carcinogenesis produced by DEN exposure. Furthermore, CGDCM and DOX therapy decreased ATP and fatty acid synthesis in rat liver cancer, which was correlated with apoptosis inhibition. CGDCM reduced cleaved caspase-3 expression in liver cancer rats when used alone or in combination with DOX, implying that apoptosis-inducing hepatic carcinogenesis was suppressed. Our results also verified the low toxicity of CGDCM injection on the internal organs of rats. Thus, this research clearly demonstrated a promising, novel anticancer approach that could be applied in future clinical studies of CGDCM and combination therapy.

Calotropis gigantea (Asclepiadaceae) (Niu jiao gua) has been used as a poultice in Chinese medicine for treating inflammatory skin diseases, e.g., neurodermatitis. This study aims to isolate the epidioxysterols from the flowers of C. gigantea, elucidate their structures and evaluate their possible inhibitory effects on the NF-κB pathway. The two epidioxysterols 9,11-dehydroergosterol peroxide (1) and ergosterol peroxide (2) were isolated from the powdered flowers of C. gigantea by ultrasonic-assisted extraction, followed by the purification of the crude extract by column chromatography (i.e., silica gel and MCI-gel CHP 20P open columns). The chemical structures of these compounds were identified through a comparison of their HRMS, 1H and 13C NMR data with those in the literature. The in vitro IKK-β inhibitory activities of compounds 1 and 2 (1–100 µM) were evaluated using an IKK α and β Assay/Inhibitor Screening Kit, which is a single-site, semi-quantitative immunoassay. Berberine was used as a positive control. The IKK-β inhibitory activities between compounds 1 and 2 were compared by a two-tailed Student’s t test to summarize the structure activity relationship. Compounds 1 and 2 exhibited a dose-dependent inhibitory activity towards IKK-β in a similar manner to that of berberine. The IKK-β inhibitory activities of these two epidioxysterols were significantly stronger (P = 0.001 for compound 1 and P = 0.028 for compound 2) than that of berberine at the concentration of 100 µM. Furthermore, at the same concentration the suppressive effect of compound 1 towards IKK-β was greater than that of compound 2 (P = 0.041), while their activities at 10 and 50 µM were comparable. The difference in the results at 100 µM therefore suggested that the double bond between C-9 and C-11 in compound 1 could be responsible for its higher inhibitory activity towards IKK-β at this concentration. 9,11-dehydroergosterol peroxide (1) and ergosterol peroxide (2) were isolated from the flowers of C. gigantea and exhibited in vitro inhibitory activities towards IKK-β.

Calotropis gigantea stem bark extract, particularly the dichloromethane fraction (CGDCM), demonstrated the most potent antiproliferative effects on hepatocellular carcinoma HepG2 and colorectal HCT116 cells. The current study focused on enhancing the effectiveness of cancer treatment with CGDCM at concentrations close to the IC50 in HCT116 cells by reducing their nutrient supply. CGDCM (2, 4, and 8 μg/mL) treatment for 24 h under glucose conditions of 4.5 g/L without fetal bovine serum (FBS) supplementation or serum starvation (G+/F−), glucose 0 g/L with 10% FBS or glucose starvation (G−/F+), and glucose 0 g/L with 0% FBS or complete starvation (G−/F−) induced a greater antiproliferative effect in HCT116 cells than therapy in complete medium with glucose 4.5 g/L and 10% FBS (G+/F+). Nonetheless, the anticancer effect of CGDCM at 4 μg/mL under (G−/F−) showed the highest activity compared to other starvation conditions. The three starvation conditions showed a significant reduction in cell viability compared to the control (G+/F+) medium group, while the inhibitory effect on cell viability did not differ significantly among the three starvation conditions. CGDCM at 4 μg/mL in (G−/F−) medium triggered apoptosis by dissipating the mitochondrial membrane potential and arresting cells in the G2/M phase. This investigation demonstrated that a decrease in intracellular ATP and fatty acid levels was associated with enhanced apoptosis by treatment with CGDCM at 4 μg/mL under (G−/F−) conditions. In addition, under (G−/F−), CGDCM at 4 μg/mL increased levels of reactive oxygen species (ROS) and was suggested to primarily trigger apoptosis in HCT116 cells. Thus, C. gigantea extracts may be useful for the future development of alternative, effective cancer treatment regimens.

The 95% ethanolic extract of the dry powder of Calotropis gigantea (C. gigantea) stem bark was separated by fractionation with different solutions to yield 4 fractions: dichloromethane (CGDCM), ethyl acetate (CGEtOAc), and water (CGW). This research focused on CGDCM-induced apoptosis in HepG2 cells with IC50 and above-IC50 values, which provide useful information for future anticancer applications. CGDCM had lower cytotoxicity on normal lung fibroblast IMR-90 cells than on HepG2 cells. Apoptotic induction of CGDCM was mediated by decreased fatty acid and ATP synthesis while increasing reactive oxygen species production. The effects of the four extracts on the activity of the four major CYP450 isoforms (CYP1A2, CYP2C9, CYP2E1 and CYP3A4) were determined using the CYP-specific model activity of each isoform. All four fractions of the extract were shown to be poor inhibitors of CYP1A2 and CYP2E1 (IC50 > 1000 μg/mL) and moderate inhibitors of CYP3A4 (IC50 = 56.54–296.9 μg/mL). CGDCM and CGW exerted moderate inhibition activities on CYP2C9 (IC50 = 59.56 and 46.38 μg/mL, respectively), but CGEtOH and CGEtOAc exhibited strong inhibition activities (IC50 = 12.11 and 20.43 μg/mL, respectively). It is proposed that C. gigantea extracts at high doses have potential for further studies to develop alternative anticancer applications. Inhibiting CYP2C9 activity may also lead to drug-herb interactions.