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.

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.

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.

Coix lacryma-jobi, a highly regarded Asian herb widely used in traditional Chinese medicine, is recognized for its dual benefits in promoting overall health and treating various diseases. While it exhibits moderate anticancer efficacy when used alone, this study investigated the enhanced anticancer potential of raw and cooked Coix lacryma-jobi var. lacryma-jobi (CL) seed extracts in combination with sorafenib against HCT116 and HepG2 cancer cell lines. The combination of sorafenib with other anticancer agents, including natural extracts, has garnered significant attention as a promising strategy for developing more effective cancer therapies. Dry powders of raw (R) and cooked (C) CL seeds, obtained from a local commercial source in Thailand, were extracted and fractionated using ethanol (E), dichloromethane (D), ethyl acetate (A), and water (W) to produce eight fractions: CLRE, CLCE, CLRD, CLCD, CLRA, CLCA, CLRW, and CLCW. The coixol content in raw and cooked seed extracts was quantified and expressed as μg of coixol per gram of extract. The cytotoxic effects of these fractions were evaluated against HCT116 and HepG2 cells using the MTT assay. Fractions demonstrating the most significant cytotoxic responses were combined with sorafenib to evaluate their synergistic effects. Apoptosis induction and mitochondrial membrane potential (MMP) were assessed, and the underlying mechanism of apoptosis was explored by analyzing reactive oxygen species (ROS) generation and antioxidant protein expression levels. Additionally, the combination treatment's effect on the phosphatidylinositol-3 kinase (PI3K)/protein kinase B (AKT)/mechanistic target of rapamycin (mTOR) pathway was investigated. One gram of CLCE and CLCD extracts contained higher coixol levels (7.02 μg and 9.69 μg, respectively) compared to CLRE and CLRD (2.66 μg and 5.96 μg, respectively). Coixol content in CLRA, CLRW, and CLCW fractions was undetectable under the study conditions. All extract fractions exhibited IC₅₀ values exceeding 1 mg/mL after 24- and 48-hour incubations with HCT116 and HepG2 cells, indicating limited cytotoxicity when used independently. CLRD and CLCD fractions were selected for combination studies at a concentration of 1 mg/mL, combined with sub-IC₅₀ concentrations of sorafenib to minimize its side effects. This combination significantly increased cytotoxicity, inducing apoptosis in HCT116 and HepG2 cells by elevating ROS levels and reducing the expression of superoxide dismutase 2 and catalase. Furthermore, the combination treatment downregulated the PI3K/AKT/mTOR pathway, indicating a targeted anticancer mechanism. The combination of CLCD with sorafenib demonstrates significant potential as a strategy for future anticancer therapies. This CL seed extract, cultivated and commercially available in Thailand, shows promise as a natural supplement to enhance the efficacy of chemotherapy in upcoming clinical anticancer applications.

The macadamia (Macadamia integrifolia Maiden & Betche, Proteaceae Family) leaves and husks were the by-products from macadamia nut production which have few reports on the phytochemicals and pharmacological activities. This study aimed to investigate the possibility of using these by-products in drug discovery by examining their phytochemicals contents, as well as their anti-colon cancer activities. Dry macadamia leaves and husks were extracted and fractionated to obtain the crude extracts (EtOH), dichloromethane (DCM), ethyl acetate (EtOAc) and water fractions. Colorimetric methods were used to determine the total amounts of phytochemicals. The anti-colon cancer activities of the extracts against human colorectal carcinoma (HCT116) cell line were assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The results showed that each gram of the samples contained total phenolics between 2.9-12.9 mg equivalent to gallic acid, total flavonoids 30.1-89.7 mg equivalent to rutin, and total triterpenoids 228.4-640.9 mg equivalent to ursolic acid. The DCM and EtOAc fractions of the husks exhibited the highest anti-colon cancer activity, with half maximal inhibitory concentrations (IC50) of 39.1 ± 4.3 and 56.9 ± 15.0 µg/mL, respectively. The EtOAc and water fractions of the leaves had IC50 values of 130.9 ± 17.4 and 127.8 ± 7.9 µg/mL, respectively. The IC50 values of other samples were greater than 200 µg/mL, while the IC50 of sorafenib, a positive control, was 5.95 ± 1.26 µg/mL. In conclusion, the leaves and husks of macadamia contained high amounts of several phytochemicals and exhibited anti-colon cancer activity against HCT116. These findings suggested that the extracts from these wastes may have potential in the discovery of anticancer agent research. Keywords: Macadamia integrifolia, Leaf, Husk, Colon cancer, Phytochemical