Dietary flavonoids: Bioavailability and biosynthesis

Flavonoids are one of the largest groups of natural plant products and are found in most, if not all, fruits and vegetables. As dietary components, flavonoids have widespread biological properties and have been associated with several health benefits, including reduced risk of cancer and cardiovascular disease. The flavonol, quercetin, one of the most ubiquitous dietary flavonoids, is found principally as glycoside conjugates in plants and quercetin-3-glucosylrhamnoside (rutin) is one of the more common forms. To establish the role of rutin as a protective agent in vivo, it is critical to understand the chemical nature of the absorbed forms and their in vivo concentrations in the circulatory and excretory system. The bioavailability of rutin is not very well understood as reflected by the varying peak plasma concentrations (Cmax) and the time taken to reach the peak plasma concentration (Tmax) reported in the literature. This may, in part, be due to the analytical techniques used which include acid or glucuronidase/sulphatase treatments to release the parent aglycone prior to quantitative analysis by low resolution, isocratic reverse-phase HPLC. In addition, there has been some debate on the types of rutin catabolites produced as a result of colonic breakdown. There is therefore a need for further more detailed information on the absoiption, metabolism and bioavailability of rutin. The underlying objective in Chapter 3 was to investigate the metabolism and absoiption of rutin in vivo after ingestion of tomato juice supplemented with 164 ?moles (l00mg) of rutin. Rutin was found to be extensively metabolised and made bioavailable to humans reflected in the identification of many phase II metabolites and catabolites. To investigate the role of colonic microflora in the breakdown of rutin, an in vitro faecal fermentation study was carried out as discussed in Chapter 4. Accumulation of quercetin in the fermentation vessel as early as 4 h after incubation indicated that deglycosylation was the initial step in the colonic breakdown of rutin. The addition of glucose to the fermentation media enhanced the deglycosylation process by almost 20 h. The study in Chapter 5 aimed at achieving this objective. An attempt was made to direct the synthesis of genistein in arabidopis and in tomato by introducing isoflavone synthase (IFS), which is the key enzyme for the entry into the isoflavonoid biosynthetic pathway. The full length IFS cDNA from soybean roots was isolated and cloned into binary vectors, pGlasglow and pCHF3 habouring the CaMV 35S promoter and into pER8 vector carrying an estrogen inducible G10-90 promoter. Using agrobacterium mediated transformation, the IFS gene was introduced in arabidopsis and tomato. Due to the nature of the pER8 construct and its G10-90 promoter, a 3-10 fold higher gene expression level was observed in arabidopsis transgenic plants transformed by pER8 binary vector than those transformed with pGlasglow. High levels of gene silencing was observed when using the CaMV 35S in arabidopsis and total gene silencing was observed when the same promoter was used in tomatoes. HPLC-PDA-MS2 analysis of leaf extracts in the highly and moderately expressed IFS arabidopsis lines failed to detect the presence of genistein. Western blot analysis implied that IFS proteins were synthesized and accumulated in the leaves of arabidopsis and were present 'freely' in the cytoplasm rather than being membrane bound as they would in their natural environment. It was, therefore, hypothesised that the 'free' IFS was not able to access the substrate, naringenin which may be compartmentalized in a pre-existing multienzyme complex, and as a consequence, IFS was unable to synthesize genistein. (Abstract shortened by ProQuest.)

Pressurized hot water extraction of hydrosable tannins from Phyllanthus tenellus Roxb.

Abstract(#br)Background(#br)Safety, environmental and economic setbacks are driving industries to find greener approaches to extract bioactive compounds from natural resources. Pressurized hot water extraction (PHWE) is among the solvent free and efficient methods for extracting bioactive compounds. Experimental(#br)In this study, the suitability of PHWE for extracting bioactive compounds such as phenolics, hydrolysable tannins and flavonoids from Phyllanthus tenellus was investigated by UPLC-qTOF-MS. Results(#br)Solvent properties of water are significantly increased through imposing temperature at 121 °C and pressure at 15 p.s.i. Pressurized hot water extraction obtained 991-folds higher hydrolysable tannins than methanol extraction. Conclusion(#br)The extraction yields of hydrolysable..

Erwinia mallotivora sp., a New Pathogen of Papaya (Carica papaya) in Peninsular Malaysia

Erwinia mallotivora was isolated from papaya infected with dieback disease showing the typical symptoms of greasy, water-soaked lesions and spots on leaves. Phylogenetic analysis of 16S rRNA gene sequences showed that the strain belonged to the genus Erwinia and was united in a monophyletic group with E. mallotivora DSM 4565 (AJ233414). Earlier studies had indicated that the causal agent for this disease was E. papayae. However, our current studies, through Koch’s postulate, have confirmed that papaya dieback disease is caused by E. mallotivora. To our knowledge, this is the first new discovery of E. mallotivora as a causal agent of papaya dieback disease in Peninsular Malaysia. Previous reports have suggested that E. mallotivora causes leaf spot in Mallotus japonicus. However, this research confirms it also to be pathogenic to Carica papaya

Antimetastatic Effects of Phyllanthus on Human Lung (A549) and Breast (MCF-7) Cancer Cell Lines

BACKGROUND: Current chemotherapeutic drugs kill cancer cells mainly by inducing apoptosis. However, they become ineffective once cancer cell has the ability to metastasize, hence the poor prognosis and high mortality rate. Therefore, the purpose of this study was to evaluate the antimetastatic potential of Phyllanthus (P. niruri, P. urinaria, P. watsonii, and P. amarus) on lung and breast carcinoma cells. METHODOLOGY/PRINCIPAL FINDINGS: Cytotoxicity of Phyllanthus plant extracts were first screened using the MTS reduction assay. They were shown to inhibit MCF-7 (breast carcinoma) and A549 (lung carcinoma) cells growth with IC(50) values ranging from 50-180 µg/ml and 65-470 µg/ml for methanolic and aqueous extracts respectively. In comparison, they have lower toxicity on normal cells with the cell viability percentage remaining above 50% when treated up to 1000 µg/ml for both extracts. After determining the non-toxic effective dose, several antimetastasis assays were carried out and Phyllanthus extracts were shown to effectively reduce invasion, migration, and adhesion of both MCF-7 and A549 cells in a dose-dependent manner, at concentrations ranging from 20-200 µg/ml for methanolic extracts and 50-500 µg/ml for aqueous extracts. This was followed by an evaluation of the possible modes of cell death that occurred along with the antimetastatic activity. Phyllanthus was shown to be capable of inducing apoptosis in conjunction with its antimetastastic action, with more than three fold increase of caspases-3 and -7, the presence of DNA-fragmentation and TUNEL-positive cells. The ability of Phyllanthus to exert antimetastatic activities is mostly associated to the presence of polyphenol compounds in its extracts. CONCLUSIONS/SIGNIFICANCE: The presence of polyphenol compounds in the Phyllanthus plant is critically important in the inhibition of the invasion, migration, and adhesion of cancer cells, along with the involvement of apoptosis induction. Hence, Phyllanthus could be a valuable candidate in the treatment of metastatic cancers

Phyllanthus spp. Induces Selective Growth Inhibition of PC-3 and MeWo Human Cancer Cells through Modulation of Cell Cycle and Induction of Apoptosis

BACKGROUND: Phyllanthus is a traditional medicinal plant that has been used in the treatment of many diseases including hepatitis and diabetes. The main aim of the present work was to investigate the potential cytotoxic effects of aqueous and methanolic extracts of four Phyllanthus species (P.amarus, P.niruri, P.urinaria and P.watsonii) against skin melanoma and prostate cancer cells. METHODOLOGY/PRINCIPAL FINDINGS: Phyllanthus plant appears to possess cytotoxic properties with half-maximal inhibitory concentration (IC(50)) values of 150-300 µg/ml for aqueous extract and 50-150 µg/ml for methanolic extract that were determined using the MTS reduction assay. In comparison, the plant extracts did not show any significant cytotoxicity on normal human skin (CCD-1127Sk) and prostate (RWPE-1) cells. The extracts appeared to act by causing the formation of a clear "ladder" fragmentation of apoptotic DNA on agarose gel, displayed TUNEL-positive cells with an elevation of caspase-3 and -7 activities. The Lactate Dehydrogenase (LDH) level was lower than 15% in Phyllanthus treated-cancer cells. These indicate that Phyllanthus extracts have the ability to induce apoptosis with minimal necrotic effects. Furthermore, cell cycle analysis revealed that Phyllanthus induced a Go/G1-phase arrest on PC-3 cells and a S-phase arrest on MeWo cells and these were accompanied by accumulation of cells in the Sub-G1 (apoptosis) phase. The cytotoxic properties may be due to the presence of polyphenol compounds such as ellagitannins, gallotannins, flavonoids and phenolic acids found both in the water and methanol extract of the plants. CONCLUSIONS/SIGNIFICANCE: Phyllanthus plant exerts its growth inhibition effect in a selective manner towards cancer cells through the modulation of cell cycle and induction of apoptosis via caspases activation in melanoma and prostate cancer cells. Hence, Phyllanthus may be sourced for the development of a potent apoptosis-inducing anticancer agent

Phyllanthusspp. Exerts Anti-Angiogenic and Anti-Metastatic Effects Through Inhibition on Matrix Metalloproteinase Enzymes

Tumor angiogenesis and metastasis are the major causes for high morbidity and mortality rates in cancer patient. Modulation on tumor angiogenesis and metastasis provides opportunities to halt progression of cancer. From our previous findings, Phyllanthus plant possesses antiproliferative effects on melanoma and prostate cancer cell lines and induction of apoptosis. The main aims of the present work were further investigated on the antimetastatic and antiangiogenic effects on cancer cells (MeWo and PC-3) and human umbilical vein endothelial cells (HUVECs) of 4 Phyllanthus species (P.amarus, P.niruri, P.urinaria and P.watsonii). Phyllanthus extracts significantly inhibited cell adhesion, migration, invasion, and transendothelial migration activities of cancer (MeWo and PC-3) cells in a dose-dependent manner (P < 0.05) by cell-matrix adhesion, Transwell migration, invasion, and transendothelial migration assays. Phyllanthus extracts were exhibited low cytotoxicity on HUVECs up to a concentration of 500.0 g/ml by MTS reduction assay. Phyllanthus extracts also exhibited antiangiogenic effects through inhibition of migration, invasion, and microcapillary like-tube structure formation in HUVECs. These observations were due to alteration in activities of matrix metalloproteinase (MMP) -2, -7, -9, and -26 in treated-endothelial and cancer cells by zymographies. These findings suggest that Phyllanthus plant has the potential to inhibit tumour metastasis and angiogenesis through the suppression of MMP enzymes

<i>Phyllanthus</i> extracts induce caspase-3 and -7 activation.

<p>The graph shows the levels of caspase-3 and -7 in treated and untreated groups of MeWo and PC-3 cells. <b>Bars show the mean ± SE</b></p

Induction of Apoptosis.

<p>TUNEL analysis of MeWo and PC-3 cancer cells after being treated with <i>Phyllanthus</i> extracts at 100× magnification. TUNEL-positive (apoptotic) cells were observable as brown stained cells (red arrow) in <i>Phyllanthus</i> extracts-treated <b>A</b>) MeWo and <b>B</b>) PC-3 cells and normal viable cells stain as blue colour. <b>C</b>) The graph shows the percentage of apoptotic index (%) of untreated and treated (<i>Phyllanthus</i> extracts and anticancer drugs) MeWo and PC-3 cancer cells from TUNEL analysis.</p

IC₅₀ values of <i>Phyllanthus</i> extracts and standard anticancer drugs on both human cancer and normal cell lines.

<p>IC₅₀ values of <i>Phyllanthus</i> extracts and standard anticancer drugs on both human cancer and normal cell lines.</p