Synthesis of Bioactive Diarylheptanoids from Alpinia officinarum and Their Mechanism of Action for Anticancer Properties in Breast Cancer Cells.

An efficient synthesis of the Alpinia officinarum-derived diarylheptanoids, viz., enantiomers of a ß-hydroxyketone (1) and an α,ß-unsaturated ketone (2) was developed starting from commercially available eugenol. Among these, compound 2 showed a superior antiproliferative effect against human breast adenocarcinoma MCF-7 cells. Besides reducing clonogenic cell survival, compound 2 dose-dependently increased the sub G1 cell population and arrested the G2-phase of the cell cycle, as revealed by flow cytometry. Mechanistically, compound 2 acts as an intracellular pro-oxidant by generating copious amounts of reactive oxygen species. Compound 2 also induced both loss of mitochondrial membrane potential (MMP) as well as lysosomal membrane permeabilization (LMP) in the MCF-7 cells. The impaired mitochondrial and lysosomal functions due to reactive oxygen species (ROS)-generation by compound 2 may contribute to its apoptotic property.

Membrane damage precedes DNA damage in hydroxychavicol treated E. coli cells and facilitates cooperativity with hydrophobic antibiotics.

Hydroxychavicol (HC), found abundantly in Piper betle leaves is credited with antimicrobial property. Previously we had shown HC induces reactive oxygen species mediated DNA damage in bacterial cells. HC also resulted in membrane compromise revealing its pleiotropic effects on cellular targets. The kinetics and exact sequence of events leading to inhibition of growth and cell death in E. coli after HC treatment remains poorly understood. We show that sub-lethal concentration (125 µg/mL) of HC causes cellular filamentation within 1 h of treatment, while a higher concentration (750 µg/mL) induces cell breakage. HC-treated cells were found to experience oxidative stress as early as 10 min, while evidence of membrane damage was apparent at 30 min. DNA damage repair genes were found to be activated at 60 min. Interestingly, HC-induced cell permeabilization was inhibited and enhanced by external Mg2+ and EDTA, respectively, suggesting that HC damages the outer membrane. Kinetic experiments revealed that HC-treated cells underwent oxidative stress, membrane damage and DNA damage in that order. Because gram negative bacteria such as E. coli are refractory to several antibiotics due to the presence of the outer membrane, we hypothesized that HC pretreatment would sensitize E. coli to hydrophobic antibiotics. Our study reveals for the first time that HC could sensitize bacteria to clinically used antibiotics due to its outer membrane damaging property.

Hydroxychavicol, a key ingredient of Piper betle induces bacterial cell death by DNA damage and inhibition of cell division.

Antibiotic resistance is a global problem and there is an urgent need to augment the arsenal against pathogenic bacteria. The emergence of different drug resistant bacteria is threatening human lives to be pushed towards the pre-antibiotic era. Botanical sources remain a vital source of diverse organic molecules that possess antibacterial property as well as augment existing antibacterial molecules. Piper betle, a climber, is widely used in south and south-east Asia whose leaves and nuts are consumed regularly. Hydroxychavicol (HC) isolated from Piper betle has been reported to possess antibacterial activity. It is currently not clear how the antibacterial activity of HC is manifested. In this investigation we show HC generates superoxide in E. coli cells. Antioxidants protected E. coli against HC induced cell death while gshA mutant was more sensitive to HC than wild type. DNA damage repair deficient mutants are hypersensitive to HC and HC induces the expression of DNA damage repair genes that repair oxidative DNA damage. HC treated E. coli cells are inhibited from growth and undergo DNA condensation. In vitro HC binds to DNA and cleaves it in presence of copper. Our data strongly indicates HC mediates bacterial cell death by ROS generation and DNA damage. Damage to iron sulfur proteins in the cells contribute to amplification of oxidative stress initiated by HC. Further HC is active against a number of Gram negative bacteria isolated from patients with a wide range of clinical symptoms and varied antibiotic resistance profiles.

Anti-inflammatory effect of allylpyrocatechol in LPS-induced macrophages is mediated by suppression of iNOS and COX-2 via the NF-kappaB pathway.

The crude ethanol extract of Piper betle leaf is reported to possess anti-inflammatory activity which has been suggested to be mediated by allylpyrocatechol (APC). In the present study, we have demonstrated the anti-inflammatory effects of APC (10 mg/kg, p.o.) in an animal model of inflammation. To investigate the mechanism(s) of this anti-inflammatory activity, we examined its effects on the lipopolysaccaride (LPS)-induced production of NO and PGE(2) in a murine macrophage cell line, RAW 264.7. APC inhibited production of NO and PGE(2) in a dose dependent manner as also decreased mRNA expression of iNOS, COX-2, IL-12p40 and TNF-alpha. Since nuclear factor-kappaB (NF-kappaB) appears to play a central role in transcriptional regulation of these proteins, we investigated the effects of APC on this transcription factor. APC inhibited LPS induced nuclear factor-kappaB (NF-kappaB) activation, by preventing degradation of the inhibitor kappaB (IkappaB). Taken together, our data indicates that APC targets the inflammatory response of macrophages via inhibition of iNOS, COX-2 and IL-12 p40 through down regulation of the NF-kappaB pathway, indicating that APC may have therapeutic potential in inflammation associated disorders.

Antioxidant activity of piper betel leaf extract and its constituents.

The 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay of the ethanol extracts of three varieties (Bangla, sweet, and Mysore) of Piper betel (pan) revealed the Bangla variety to possess the best antioxidant activity that can be correlated with the total phenolic content and reducing powers of the respective extracts. Column chromatography of the extract of the Bangla variety led to the isolation of chevibetol (CHV), allylpyrocatechol (APC), and their respective glucosides. The HPTLC analyses of the extracts revealed similar chemical profiles in all three P. betel varieties, although the concentrations of CHV and APC were significantly less in the sweet and Mysore varieties. Among the isolated compounds, APC showed the best results in all the in vitro experiments. It could prevent Fe(II)-induced lipid peroxidation (LPO) of liposomes and rat brain homogenates as well as gamma-ray-induced damage of pBR322 plasmid DNA more efficiently than CHV. The superior anti-LPO and radioprotective activities of APC vis-à-vis those of CHV could not be explained by their respective Fe(II) chelation and .OH radical scavenging capacities. The better ability of APC to scavenge O2-. radicals and H2O2 might account for the results.