Evangeline C. Amor

Tannin-rich fraction from Terminalia catappa inhibits quorum sensing (QS) in Chromobacterium violaceum and the QS-controlled biofilm maturation and LasA staphylolytic activity in Pseudomonas aeruginosa

AIM OF THE STUDY The study aimed to test the activity of Terminalia catappa L. against bacterial quorum sensing (QS) in order to provide a potential scientific basis for the traditional use of leaf extracts of this plant as an antiseptic. MATERIALS AND METHODS The anti-QS activity of the methanolic leaf extract of Terminalia catappa was detected through the inhibition of the QS-controlled violacein pigment production in Chromobacterium violaceum. Fractions resulting from size-exclusion chromatography were assayed. The most active fraction was characterized through qualitative phytochemical detection methods. The effect of this fraction on known QS-controlled phenotypes in test strains was assessed. RESULTS The fraction with the highest activity (labeled as TCF12) was characterized to be tannin-rich. It specifically inhibited QS-controlled violacein production in Chromobacterium violaceum with 50% reduction achieved at 62.5 μg mL(-1) without significantly affecting growth up to about 962 μg mL(-1). The assessment of its effects on LasA activity of Pseudomonas aeruginosa ATCC 10145 found that the production of this virulence determinant is reduced in a concentration dependent manner with about 50% reduction at 62.5 μg mL(-1). Furthermore, it was found that TCF12 was able to inhibit the maturation of biofilms of Pseudomonas aeruginosa, a phenotype that has also been known to be QS-regulated. CONCLUSION Therefore, tannin-rich components of Terminalia catappa leaves are able to inhibit certain phenotypic expression of QS in the test strains used.

Induction of autophagy by dimethyl cardamonin is associated with proliferative arrest in human colorectal carcinoma HCT116 and LOVO cells

Dimethyl cardamonin (2′,4′‐dihydroxy‐6′‐methoxy‐3′,5′‐dimethylchalcone; DMC) is a naturally occurring chalcone, and it is the major compound isolated from the leaves of Syzygium samarangense (Blume) Merr. & L.M. Perry (Myrtaceae). Experiments were conducted to determine the effects of DMC on cell proliferation, cell‐cycle distribution, and programmed cell death in cultures of human colorectal carcinoma HCT116 and LOVO cells. Results showed that DMC inhibited HCT116 and LOVO cell proliferation and induced G2/M cell cycle arrest, which was associated with the conversion of microtubule associated protein light chain 3 (LC3)‐I–LC3‐II, an autophagosome marker, and the incorporation of monodansylcadaverine (MDC), a marker for the acidic compartment of autolysosomes or acidic vesicular organelles. The treatment of HCT116 and LOVO cells using a combination of DMC with an autophagy inhibitor, such as 3‐methyladenine (3‐MA), beclin 1 siRNA, or atg5 siRNA, suppressed the effect of DMC‐mediated anti‐proliferation. These results imply that DMC can suppress colorectal carcinoma HCT116 and LOVO cell proliferation through a G2/M phase cell‐cycle delay, and can induce autophagy, the hallmark of Type II programmed cell death (PCD). Taken together, our results suggest that DMC may be an effective chemotherapeutic agent for HCT116 and LOVO colorectal carcinoma cells. J. Cell. Biochem. 112: 2471–2479, 2011.

Cytotoxic C-Methylated Chalcones from Syzygium samarangense.

Abstract The flavonoids 2′-hydroxy-4′,6′-dimethoxy-3′-methylchalcone (1), 2′,4′-dihydroxy-6′-methoxy-3′,5′-dimethylchalcone (2), 2′,4′-dihydroxy-6′-methoxy-3′-methylchalcone (3), 2′,4′-dihydroxy-6′-methoxy-3′-methyldihydrochalcone (4) and 2′,4′-dihydroxy-6′-methoxy-3′,5′-dimethyldihydrochalcone (5), isolated from Syzygium samarangense. (Blume) Merr. & L.M. Perry (Myrtaceae), were subjected to cytotoxicity testing using the dimethylthiazoldiphenyl tetrazolium (MTT) assay. The cell lines used were the Chinese hamster ovarian (CHO-AA8) and the human mammary adenocarcinoma, (MCF-7 and SKBR-3). Among the test compounds, 2 exhibited significant differential cytotoxicity against the MCF-7 cell line with an IC50 of 0.0015 ± 0.0001 nM. It was also cytotoxic against the SKBR-3 cell line with an IC50 of 0.0128 ± 0.0006 nM. Doxorubicin, the positive control, had an IC50 of 2.60 ± 0.28 × 10−4 nM against the MCF-7 cell line and an IC50 of 2.76 ± 0.52 × 10−5 nM against the SKBR-3 cell line. When tested in a mechanism-based yeast bioassay for detecting DNA-damaging agents using genetically engineered Saccharomyces cerevisiae. RS322Y (RAD52) mutant strain and (LF15/11) (RAD+) wild-type strain, 2 showed significant selective cytotoxicity against the RAD52 yeast mutant strain. It had an IC12 of 0.1482 nM, as compared with the positive control, streptonigrin, which had an IC12 of 0.0134 nM. Hence, 2 is a cytotoxic natural product with potential anticancer application.

Prolyl endopeptidase inhibitors from Syzygium samarangense (Blume) Merr. & L. M. Perry.

Compounds isolated from the hexane extract of the leaves of Syzygium samarangense (Blume) Merr. & L. M. Perry were tested for inhibitory activity against the following serine proteases: trypsin, thrombin and prolyl endopeptidase. The compounds were identified as an intractable mixture of α-carotene and β-carotene (1), lupeol (2), betulin (3), epi-betulinic acid (4), 2′,4′-dihydroxy-6′-methoxy-3′-methylchalcone (5), 2′-hydroxy-4′,6′-dimethoxy-3′- methylchalcone (6), 2′,4′-dihydroxy-6′-methoxy-3′,5v-dimethylchalcone (7), 2′,4′-dihydroxy- 6′-methoxy-3′-methyldihydrochalcone (8) and 7-hydroxy-5-methoxy-6,8-dimethylflavanone (9). Hydrogenation of compounds 5, 6 and 7 yielded compound 8, 2′-hydroxy-4′,6′-dimethoxy- 3′-methyldihydrochalcone (10) and 2′,4′-dihydroxy-6′-methoxy-3′,5-dimethyldihydrochalcone (11), respectively. The hydrogenated products of compounds 6 and 7 were also tested for enzyme inhibitory activity. In addition, β-sitosterol (12) and β-ᴅ-sitosterylglucoside (13) were also isolated. This is the first report of the isolation of compounds 1-6, 8 and 13 from this plant. Compounds 3-8 and 10 exhibited significant and selective inhibition against prolyl endopeptidase among three serine proteases. This is the first report of this kind of activity for all these compounds.

Stercurensin inhibits nuclear factor-κB-dependent inflammatory signals through attenuation of TAK1–TAB1 complex formation

We identified a chalcone, 2′,4′‐dihydroxy‐6′‐methoxy‐3′‐methylchalcone (stercurensin), as an active compound isolated from the leaves of Syzygium samarangense. In the present study, the anti‐inflammatory effects and underlying mechanisms of stercurensin were examined using lipopolysaccharide (LPS)‐stimulated RAW264.7 cells and mice. To determine the effects of stercurensin in vitro, inducible nitric oxide synthase (iNOS) and cyclooxygenase‐2 (COX‐2) expression were analyzed by RT‐PCR and immunoblotting. Nuclear factor‐κB (NF‐κB) activation and its upstream signaling cascades were also investigated using a dual‐luciferase reporter assay, electrophoretic mobility shift assay, immunoblotting, immunofluorescence, and immunoprecipitation. To verify the effects of stercurensin in vivo, the mRNA expression levels of iNOS and COX‐2 were evaluated in isolated mouse peritoneal macrophages by quantitative real‐time PCR, and the production of tumor necrosis factor‐α (TNF‐α), interleukin‐6 (IL‐6), and IL‐1β were assessed in serum samples from mice using a Luminex system. Pretreatment with stercurensin reduced LPS‐induced iNOS and COX‐2 expression, thereby inhibiting nitric oxide (NO) and prostaglandin E2 production, respectively. In addition, an inhibitory effect of stercurensin on NF‐κB activation was shown by the recovery of LPS‐induced inhibitor of κB (I‐κB) degradation after blocking the transforming growth factor‐β‐activated kinase 1 (TAK1)/I‐κB kinase signaling pathway. In mouse models, stercurensin negatively regulated NF‐κB‐dependent pro‐inflammatory mediators and cytokines. These results demonstrate that stercurensin modulates NF‐κB‐dependent inflammatory pathways through the attenuation of TAK1–TAB1 complex formation. Our findings demonstrating the anti‐inflammatory effects of stercurensin in vitro and in vivo will aid in understanding the pharmacology and mode of action of stercurensin. J. Cell. Biochem. 112: 548–558, 2011.

Spasmolytic Flavonoids from Syzygium samarangense (Blume) Merr. & L.M. Perry

The hexane extract of Syzygium samarangense (Ss.Hex) dose-dependently (10D1000 μg/ ml) relaxed the spontaneously contracting isolated rabbit jejunum. Four rare C-methylated flavonoids with a chalcone and a flavanone skeleton were isolated from Ss.Hex and were subsequently tested for spasmolytic activity. All flavonoids, identified as 2′-hydroxy-4′,6′- dimethoxy-3′-methylchalcone (1), 2′,4′-dihydroxy-6′-methoxy-3′,5′-dimethylchalcone (2), 2′,4′-dihydroxy-6′-methoxy-3′-methylchalcone (3), and 7-hydroxy-5-methoxy-6,8-dimethylflavanone (4), showed dose-dependent spasmolytic activity in the rabbit jejunum with IC50 values of 148.3 ± 69.4, 77.2 ± 43.5, 142.4 ± 58.6 and 178.5 ± 37.5 μg/ml (mean ± SEM), respectively. The dihydrochalcone derivative of compound 1, 2′-hydroxy-4′,6′-dimethoxy-3′- methyldihydrochalcone (5), when tested for spasmolytic activity, did not significantly relax the smooth muscle relative to the other compounds. Verapamil, a standard spasmolytic, has an IC50 value of 0.16 ± 0.04 μg/ml. This is the first report of the relaxant activity of chalcones, specifically of compounds 1-3.

Inhibitory effects of aurentiacin from Syzygium samarangense on lipopolysaccharide-induced inflammatory response in mouse macrophages

Aurentiacin is a chalcone isolated from Syzygium samarangense. In the present study, we examined the anti-inflammatory effects of aurentiacin in lipopolysaccharide (LPS)-stimulated mouse macrophages. Aurentiacin significantly inhibited LPS-induced nitric oxide (NO) production in RAW264.7 cells concomitantly with the suppression of inducible nitric oxide synthase (iNOS) expression. Aurentiacin also reduced the mRNA levels of pro-inflammatory cytokines such as tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6). Electrophoretic mobility shift assays (EMSAs) and reporter gene assays indicated that DNA binding and transcriptional activities of nuclear factor-κB (NF-κB)/p65 were decreased by aurentiacin in LPS-stimulated RAW264.7 cells. Moreover, results from chromatin immunoprecipitation (ChIP) assays over the promoter region of the iNOS gene were in agreement with the EMSA results. Pretreatment with aurentiacin prevented the nuclear translocation of p65 by blocking the phosphorylation of I-κB kinase (IKK). Aurentiacin also attenuated the phosphorylation (Ser536) and acetylation (Lys310) of p65 and phosphorylation of MAPKs. In an inflammatory animal model, the intraperitoneal (i.p.) injection of aurentiacin suppressed the release of pro-inflammatory cytokines. Moreover, the level of iNOS protein ex vivo was decreased by aurentiacin similar to the result in vitro. Taken together, these results suggest that aurentiacin shows anti-inflammatory activity related to the inhibition of NF-κB activation.

Analgesic activity of extracts of Kyllinga monocephala.

The analgesic activity of the methanol extract of the leaves of Kyllinga monocephala Rottb. (Cyperaceae) was evaluated using the acetic acid-induced writhing test on mice and was found to significantly reduce the number of writhes in mice by half. Following a bioassay-guided fractionation scheme, statistically significant analgesic activity was observed with both the hexane and ethyl acetate partitions. An active fraction was identified after fractionation of the hexane extract, whereas two active fractions were identified after fractionation of the ethyl acetate extract. This is the first report of the analgesic activity of K. monocephala.