Induction of apoptosis in HeLa cells via caspase activation by resveratrol and genistein.

Selectively inducing apoptosis in cancer cells is a much desired strategy when tolerance toward side effects is minimal during chemotherapy. In our search for natural products that can induce apoptosis in human cervical cancer cells (HeLa), we selected resveratrol and genistein for our study. We conducted several experiments to test whether genistein can synergistically enhance the apoptotic potential of resveratrol at doses lower than the usual cytotoxic dose. Both resveratrol and genistein were able to induce apoptosis by enhancing the activities of caspase-9 and caspase-3 by themselves and also in combination. After 24 h of exposure to resveratrol and genistein, individually or in combination, lowered mitochondrial membrane potential was observed in HeLa cells. In addition, the mitochondrial membrane potential in HeLa cells was decreased, forcing JC-1 to stay in the monomeric form. The monomeric JC-1(5,5',6,6' -tetrachloro-1,1',3,3'-tetraethyl benzimedazolyl carbocyanine iodide) emitted green fluorescence. In the control group, the color of the fluorescence was red due to aggregation of JC-1 in the physiological pH. The treatment groups exhibited DNA fragmentation as the hallmark of apoptotic nuclear features. We also detected an obvious decrease in the level of HDM2 gene expression after both individual and combination treatments with resveratrol and genistein. Our findings suggest that resveratrol and genistein when combined can induce apoptosis at doses lower than usual doses, through the activation of caspases cascade, and by decreasing the expression of HDM2.

Treatment of ovarian cancer cells with nutlin-3 and resveratrol combination leads to apoptosis via caspase activation.

The current study was focused on the induction of apoptotic effects of resveratrol along with the combination treatments of nutlin-3 and transforming growth factor-ß (TGF-ß) against the human ovarian cancer cell line A2780/CP70. To determine the extent of apoptosis following the above-mentioned treatments, we assessed the execution of apoptotic events that proceed via caspase activation and cytochrome c release. We estimated the caspase-3 and -9 activities using a direct enzymatic assay that measures the cleavage of synthetic peptide substrate (N-acetyl-Asp-Glu-Val-Asp-p-nitroanilide). Our experiments showed an increase in caspase-3 and -9 activities in the cells that were treated with the combination of resveratrol (5 µM) with nutlin-3 (5 µM) or TGF-ß (1 µg/mL). Since activation of procaspase-3 by caspase-9 requires the release of cytochrome c into the cytoplasm, we measured the levels of cytochrome c in the cytoplasm by western blot experiments. The data indicated a considerable increase in caspase-3 and cytochrome c levels when cells were treated with drugs for 24 hours. Experiments with 4,6'-diamino-2-phenylindole dihydrochloride (DAPI) staining also confirmed the induction of apoptosis in all the above-mentioned treatments done at 24 and 48 hours. These results support our hypothesis that resveratrol combination can induce programmed cell death at doses that are less than half of what is typically needed for nutlin-3 and TGF-ß to induce apoptosis.

Anti-diabetic properties of three common Bidens pilosa variants in Taiwan.

Bidens pilosa L. var. radiata (BPR), B. pilosa L. var. pilosa (BPP), and B. pilosa L. var. minor (BPM) are common variants of a plant often used as a folk remedy for diabetes in Taiwan. However, the three variants are often misidentified and little is known about their relative anti-diabetic efficacy and chemical composition. In this paper, we have first developed a method based on GC-MS and cluster analysis with visualization to assist in rapidly determining the taxonomy of these three Bidens variants. GC-MS was used to determine the chemical compositions of supercritical extracts, and differences and similarities in the variants were determined by hierarchical cluster analysis. Next, the HPLC profiles of the methanol crude extracts in the Bidens plants and evaluated anti-diabetic effects of methanol crude extracts were compared, as well as three polyacetylenic compounds of the Bidens plants using db/db mice. Single-dose and long-term experiments showed that the BPR extract had higher glucose-lowering and insulin-releasing activities than extracts from the other two variants, and that cytopiloyne was the most effective pure compound among the three polyacetylenic compounds. BPR extract and cytopiloyne also significantly reduced the percentage of the glycosylated hemoglobin A1c in db/db mice. Besides, both animal studies and HPLC analysis demonstrated a good correlation between anti-diabetic efficacy of the Bidens extracts and the particular polyacetylenes present.

Antioxidant and biocidal activities of Carum nigrum (seed) essential oil, oleoresin, and their selected components.

In the present study, chemical constituents of the essential oil and oleoresin of the seed from Carum nigrum obtained by hydrodistillation and Soxhlet extraction using acetone, respectively, have been studied by GC and GC-MS techniques. The major component was dillapiole (29.9%) followed by germacrene B (21.4%), beta-caryophyllene (7.8%), beta-selinene (7.1%), and nothoapiole (5.8%) along with many other components in minor amounts. Seventeen components were identified in the oleoresin (Table 2) with dillapiole as a major component (30.7%). It also contains thymol (19.1%), nothoapiole (15.2.3%), and gamma-elemene (8.0%). The antioxidant activity of both the essential oil and oleoresin was evaluated in mustard oil by monitoring peroxide, thiobarbituric acid, and total carbonyl and p-anisidine values of the oil substrate. The results showed that both the essential oil and oleoresin were able to reduce the oxidation rate of the mustard oil in the accelerated condition at 60 degrees C in comparison with synthetic antioxidants such as butylated hydroxyanisole and butylated hydroxytoluene at 0.02%. In addition, individual antioxidant assays such as linoleic acid assay, DPPH scavenging activity, reducing power, hydroxyl radical scavenging, and chelating effects have been used. The C. nigrum seed essential oil exhibited complete inhibition against Bacillus cereus and Pseudomonas aeruginosa at 2000 and 3000 ppm, respectively, by agar well diffusion method. Antifungal activity was determined against a panel of foodborne fungi such as Aspergillus niger, Penicillium purpurogenum, Penicillium madriti, Acrophialophora fusispora, Penicillium viridicatum, and Aspergillus flavus. The fruit essential oil showed 100% mycelial zone inhibition against P. purpurogenum and A. fusispora at 3000 ppm in the poison food method. Hence, both oil and oleoresin could be used as an additive in food and pharmaceutical preparations after screening.