Sugumaran Manickam

Growth inhibition of human gynecologic and colon cancer cells by Phyllanthus watsonii through apoptosis induction.

Phyllanthus watsonii Airy Shaw is an endemic plant found in Peninsular Malaysia. Although there are numerous reports on the anti cancer properties of other Phyllanthus species, published information on the cytotoxicity of P. watsonii are very limited. The present study was carried out with bioassay-guided fractionation approach to evaluate the cytotoxicity and apoptosis induction capability of the P. watsonii extracts and fractions on human gynecologic (SKOV-3 and Ca Ski) and colon (HT-29) cancer cells. P. watsonii extracts exhibited strong cytotoxicity on all the cancer cells studied with IC50 values of ≤ 20.0 µg/mL. Hexane extract of P. watsonii was further subjected to bioassay-guided fractionation and yielded 10 fractions (PW-1→PW-10). PW-4→PW-8 portrayed stronger cytotoxic activity and was further subjected to bioassay-guided fractionation and resulted with 8 sub-fractions (PPWH-1→PPWH-8). PPWH-7 possessed greatest cytotoxicity (IC50 values ranged from 0.66 – 0.83 µg/mL) and was selective on the cancer cells studied. LC-MS/MS analysis of PPWH-7 revealed the presence of ellagic acid, geranic acid, glochidone, betulin, phyllanthin and sterol glucoside. Marked morphological changes, ladder-like appearance of DNA and increment in caspase-3 activity indicating apoptosis were clearly observed in both human gynecologic and colon cancer cells treated with P. watsonii especially with PPWH-7. The study also indicated that P. watsonii extracts arrested cell cycle at different growth phases in SKOV-3, Ca Ski and HT-29 cells. Cytotoxic and apoptotic potential of the endemic P. watsonii was investigated for the first time by bioassay-guided approach. These results demonstrated that P. watsonii selectively inhibits the growth of SKOV-3, Ca Ski and HT-29 cells through apoptosis induction and cell cycle modulation. Hence, P. watsonii has the potential to be further exploited for the discovery and development of new anti cancer drugs.

Effect of extracts from Phyllanthus watsonii Airy Shaw on cell apoptosis in cultured human breast cancer MCF-7 cells.

Species of Phyllanthus have traditionally been used for hundreds of years for treating many ailments including diabetes, anemia, bronchitis and hepatitis. The present study aims to investigate the cytotoxic and apoptotic effects of methanol (PWM), hexane (PWH) and ethyl acetate (PWE) extracts from the leaves of the endemic plant Phyllanthus watsonii Airy Shaw (Phyllanthaceae) on MCF-7 human breast cancer cells. We observed that the PWM, PWH and PWE extracts were cytotoxic and selectively inhibited the growth and proliferation of MCF-7 cells compared to untreated control in a dose dependent manner with an IC(50) of 12.7 ± 4.65, 7.9 ± 0.60 and 7.7 ± 0.29 μg/ml, respectively. However, the extracts were not toxic at these concentrations to normal human lung fibroblast MRC-5 cells. Cell death induced by PWM, PWH and PWE extracts were mainly due to apoptosis which was characterized by apoptotic morphological changes and a nuclear DNA fragmentation. Caspase-3 activation following P. watsonii extracts treatment was also evident for apoptotic cell death which was preceded by an S phase cell cycle perturbation. The results suggested that the cytotoxic activity of P. watsonii extracts was related to an early event of cell cycle perturbation and a later event of apoptosis. Hence, P. watsonii displays potential to be further exploited in the discovery and development of new anticancer agents.

Holoparasitic Rafflesiaceae possess the most reduced endophytes and yet give rise to the world's largest flowers

BACKGROUND AND AIMS Species in the holoparasitic plant family Rafflesiaceae exhibit one of the most highly modified vegetative bodies in flowering plants. Apart from the flower shoot and associated bracts, the parasite is a mycelium-like endophyte living inside their grapevine hosts. This study provides a comprehensive treatment of the endophytic vegetative body for all three genera of Rafflesiaceae (Rafflesia, Rhizanthes and Sapria), and reports on the cytology and development of the endophyte, including its structural connection to the host, shedding light on the poorly understood nature of this symbiosis. METHODS Serial sectioning and staining with non-specific dyes, periodic-Schiffs reagent and aniline blue were employed in order to characterize the structure of the endophyte across a phylogenetically diverse sampling. KEY RESULTS A previously identified difference in the nuclear size between Rafflesiaceae endophytes and their hosts was used to investigate the morphology and development of the endophytic body. The endophytes generally comprise uniseriate filaments oriented radially within the host root. The emergence of the parasite from the host during floral development is arrested in some cases by an apparent host response, but otherwise vegetative growth does not appear to elicit suppression by the host. CONCLUSIONS Rafflesiaceae produce greatly reduced and modified vegetative bodies even when compared with the other holoparasitic angiosperms once grouped with Rafflesiaceae, which possess some vegetative differentiation. Based on previous studies of seeds together with these findings, it is concluded that the endophyte probably develops directly from a proembryo, and not from an embryo proper. Similarly, the flowering shoot arises directly from the undifferentiated endophyte. These filaments produce a protocorm in which a shoot apex originates endogenously by formation of a secondary morphological surface. This degree of modification to the vegetative body is exceptional within angiosperms and warrants additional investigation. Furthermore, the study highlights a mechanical isolation mechanism by which the host may defend itself from the parasite.

Floral structure and development in Rafflesiaceae with emphasis on their exceptional gynoecia.

PREMISE OF THE STUDY The holoparasitic plant family Rafflesiaceae include the worlds largest flowers. Despite their iconic status, relatively little is known about the morphology and development of their flowers. A recent study clarified the organization of the outer (sterile) floral organs, surprisingly revealing that their distinctive floral chambers arose via different developmental pathways in the two major genera of the family. Here, we expand that research to investigate the structure and development of the reproductive organs of Rafflesiaceae. METHODS Serial sectioning, scanning electron microscopy, and x-ray tomography of floral buds were employed to reconstruct the structure and development of all three Rafflesiaceae genera. KEY RESULTS Unlike most angiosperms, which form their shoot apex from the primary morphological surface, the shoot apex of Rafflesiaceae instead forms secondarily via internal cell separation (schizogeny) along the distal boundary of the host-parasite interface. Similarly, the radially directed ovarial clefts of the gynoecium forms via schizogeny within solid tissue, and no carpels are initiated from the floral apex. CONCLUSIONS The development of the shoot apex and gynoecium of Rafflesiaceae are highly unusual. Although secondary formation of the morphological surface from the shoot apex has been documented in other plant groups, secondary derivation of the inner gynoecium surface is otherwise unknown. Both features are likely synapomorphies of Rafflesiaceae. The secondary derivation of the shoot apex may protect the developing floral shoot as it emerges from within dense host tissue. The secondary formation of the ovarial clefts may generate the extensive placental area necessary to produce hundreds of thousands of ovules.

Leptospermum flavescens Constituent-LF1 Causes Cell Death through the Induction of Cell Cycle Arrest and Apoptosis in Human Lung Carcinoma Cells.

Leptospermum flavescens Sm. (Myrtaceae), locally known as ‘Senna makki’ is a smallish tree that is widespread and recorded to naturally occur in the montane regions above 900 m a.s.l from Burma to Australia. Although the species is recorded to be used traditionally to treat various ailments, there is limited data on biological and chemical investigations of L. flavescens. The aim of the present study was to investigate and understand the ability of L. flavescens in inducing cell death in lung cancer cells. The cytotoxic potentials of the extraction yields (methanol, hexane, ethyl acetate and water extracts as wells as a semi pure fraction, LF1) were evaluated against two human non-small cell lung carcinoma cell lines (A549 and NCI-H1299) using the MTT assay. LF1 showed the greatest cytotoxic effect against both cell lines with IC50 values of 7.12 ± 0.07 and 9.62 ± 0.50 μg/ml respectively. LF1 treated cells showed a sub-G1 region in the cell cycle analysis and also caused the presence of apoptotic morphologies in cells stained with acridine orange and ethidium bromide. Treatment with LF1 manifested an apoptotic population in cells that were evaluated using the Annexin V/ propidium iodide assay. Increasing dosage of LF1 caused a rise in the presence of activated caspase-3 enzymes in treated cells. Blockage of cell cycle progression was also observed in LF1-treated cells. These findings suggest that LF1 induces apoptosis and cell cycle arrest in treated lung cancer cells. Further studies are being conducted to isolate and identify the active compound as well to better understand the mechanism involved in inducing cell death.

Reading between the vines: Hosts as islands for extreme holoparasitic plants

PREMISE OF THE STUDY Partitioning of population genetic variation in plants may be affected by numerous factors including life history and dispersal characteristics. In parasitic plants, interactions with host populations may be an additional factor influencing partitioning. To test for hierarchical population genetic patterns related to obligate endoparasitism, we studied three species of Rafflesiaceae, which grow as extremely reduced endophytes infecting Tetrastigma vines in Southeast Asia. METHODS Microsatellite markers were developed and multilocus genotypes were determined for Rafflesia cantleyi, Rafflesia tuan-mudae, and Sapria himalayana and each of their Tetrastigma hosts. Relatedness among parasite individuals was estimated, and AMOVAs were used to determine levels of population genetic subdivision. KEY RESULTS Microsatellite genotypes for 340 paired parasite and host samples revealed that host vines were infected by numerous Rafflesiaceae individuals that may spread for up to 14 m within stem tissues. Surprisingly, Rafflesiaceae parasites within a given host are significantly more closely related to each other than individuals of the same species in other host individuals. The pattern of hierarchical population genetic subdivision we detected across species is likely due to limited seed dispersal with reinfection of natal host vines. CONCLUSIONS These findings demonstrate common population genetic patterns between animal and plant parasites, potentially indicating advantages of close relatives infecting hosts. This study also has important conservation implications for Rafflesiaceae since our data suggest that destruction of a single infected host vine could result in large genetic losses.

Automated plant identification using artificial neural network and support vector machine

ABSTRACT Ficus is one of the largest genera in plant kingdom reaching to about 1000 species worldwide. While taxonomic keys are available for identifying most species of Ficus, it is very difficult and time consuming for interpretation by a nonprofessional thus requires highly trained taxonomists. The purpose of the current study is to develop an efficient baseline automated system, using image processing with pattern recognition approach, to identify three species of Ficus, which have similar leaf morphology. Leaf images from three different Ficus species namely F. benjamina, F. pellucidopunctata and F. sumatrana were selected. A total of 54 leaf image samples were used in this study. Three main steps that are image pre-processing, feature extraction and recognition were carried out to develop the proposed system. Artificial neural network (ANN) and support vector machine (SVM) were the implemented recognition models. Evaluation results showed the ability of the proposed system to recognize leaf images with an accuracy of 83.3%. However, the ANN model performed slightly better using the AUC evaluation criteria. The system developed in the current study is able to classify the selected Ficus species with acceptable accuracy.