Antitumor Effect of Zerumbone Isolated from Lempoyang (Zingiber Zerumbet) on Human Cervical Cancer Cells and Mouse Cervical Intraepithelial Neoplasia

Malaysia as a tropical country is a rich source of biologically active phytochemicals, which could be useful as an alternative to the current unsafe regimens of cancer treatment. This includes the use of cisplatin (CIS), the current chemotherapeutic drug to treat cervical cancer, the second most lethal cancer affecting women in Malaysia. Therefore, anti-tumor activities of zerumbone (ZER) were investigated in both in vitro and in vivo cervical cancer models. This natural compound was isolated from the edible plant Zingiber zerumbet, locally known as Lempoyang, through column chromatography and hydrodistillation methods. The chemical structure of ZER was confirmed using NMR. The cytotoxic effects of ZER were tested in human cervical cancer cell lines (HeLa) using MTT assay and compared concurrently to cisplatin. Zerumbone’s induction of HeLa cancer cell deaths were quantified using AO/PI double staining and flow cytometry. Transmission and scanning electron microscopic analyses were done to evaluate ultra-morphological changes. The effect of ZER on caspase-3 and caspase-9 was evaluated colorimetrically in HeLa cells. The in vivo model of cervical intraepithelial neoplasia (CIN) was induced in pregnant female Balb/c mice using Diethylstilboestrol (DES). Cervical tissues were stained with hematoxylin and eosin (H&E) and viewed under light microscopy and the in vivo antiproliferative properties of ZER was confirmed by the immunohistochemical staining of proliferating cellular nuclear antigen (PCNA) as a proliferation marker and the PCNA labeling index was obtained. Apoptosis (Bcl-2 & Bax) and G2/M-cell cycle arrest (cdc25B, cyclinB1 and Chk2) associated proteins were investigated using immunohistochemistry. Moreover, RT-PCR was used to amplify mRNA of Bcl-2, Bax, c-myc and β-actin genes. The genetic material was obtained by laser capture microdissection microscopy (LCMM). No previous toxicological investigations have been carried out on this compound. Hence, acute, sub-acute and sub-chronic toxicity studies and ZER was evaluated for its behavioural, biochemical and histo-pathological effects. Findings of NMR coincide to the previously published data. However, ZER was able to exert an antiproliferative effect towards HeLa when isolated by both hydrodistillation and column chromatography, with an IC50 of 20.30±1.1 μM and 20.41±0.9 μM (p>0.05, student t-test, n=3), respectively. AO/PI-stained HeLa cells showed that ZER induced apoptosis in a time-dependent manner with insignificant statistical (p>0.05) difference in necrosis between various doses of this compound. Moreover, flow cytometric evaluation of the effect of ZER on DNA content by cell cycle phase distribution revealed that the cell populations at G0 and G2/M phases were significantly different (p0.05) serum concentrations of AST, ALP, ALT and GGT. No histopathological changes were observed in the hepatic, renal, cardiac and gastrointestinal tissues. These histomorphological findings were supported by the insignificant differences (p>0.05) between the mean lesion scores of hepatic and renal tissues. Collectively, results presented in this study demonstrated that ZER causes metaphasal blockage in HeLa cells, leading to growth inhibition and apoptosis, which was later confirmed to be through mitochondrial pathways. As ZER exhibits similar pharmacological activity to CIS, it possesses the potential to be developed as an antiproliferative agent for cervical cancer but producing less side effects, as the compound was shown to have no toxicological signs compared to the clinical complications of CIS

Tamoxifen drug loading solid lipid nanoparticles prepared by hot high pressure homogenization techniques.

As drug delivery systems Nanoparticulate widely investigated because of many advantages such as smaller size, controlled drug release potential, targeting ability, enhancement of therapeutic efficacy and reduction of toxicity. So, Solid Lipid Nanoparticles have recently received considerable attention as alternative drug delivery carrier. In this study Solid Lipid Nanoparticles (SLNs) containing tamoxifen, nonsteroidal antiestrogens have been loaded and to be used as breast cancer therapy, were prepared by hot High Pressure Homogenization techniques. Tamoxifen loaded SLNs seem to have dimensional properties useful for parenteral administration. Preliminary study of antiproliferative activity in vitro, carried out on MCF-7 cell line (human breast cancer cells), demonstrated that SLNs, containing tamoxifen showed an antitumoral activity comparable to free drug. Tamoxifen loaded SLNs seem to have dimensional properties useful for parenteral administration. SLN was characterized by Differential Scanning Calorimetery (DSC), Transmission Electron Microscopy (TEM), Zeta Potential and Particle Size. The results of characterization studies strongly support the potential application of Tamoxifen-loaded SLNs as a carrier system. The SLN presented here are well suited for certain drug delivery applications, particularly breast cancer therapy

Erectogenic Effects of Clerodendron capitatum

Clerodendron capitatum (Willd) (family: verbenaceae) is locally named as Gung and used traditionally to treat erectile dysfunction. Therefore, the current study was designed to investigate the erectogenic properties of C. capitatum. The relaxation effect of this plant was tested on phenylephrine precontracted rabbit corpus cavernosum smooth muscle (CCSM). The effects of C. capitatum were also examined on isolated Guinea pig atria alone, in the presence of calcium chloride (Ca2+ channel blocker), atropine (cholinergic blocker), and glibenclamide (ATP-sensitive K+ channel blocker). These effects were confirmed on isolated rabbit aortic strips. The extract, when tested colorimetrically for its inhibitory activities on phosphordiesterase-5 (PDE-5) in vitro towards p-nitrophenyl phenyl phosphate (PNPPP), was observed to induce significant dose-dependent inhibition of PDE-5, with an ID50 of 0.161 mg/ml (<.05). In conclusion, our results suggest that C. capitatum possesses a relaxant effect on CCSM, which is attributable to the inhibition of PDE-5, but not mediated by the release calcium, activation of adrenergic or cholinergic receptors, or the activation of potassium channels

Oncolysis of breast, liver and leukemia cancer cells using ethyl acetate and methanol extracts of goniothalamus umbrosus.

This current study, aims to investigate the ethyl acetate and methanol extracts of Goniothalamus umbrosus for their anticancer effects on several human cancer cells namely, the MCF-7 breast cancer, HT-29 colon cancer and CEM-ss leukemia cell lines using a 3 days MTT (3-(4 and 5-dimethylthiazol-2-yl)-2 and 5-diphenyltetrazolium bromide) assay. Morphological changes and probable mode of cancer cell death induced by bioactive G. umbrosus extract were examined. DNA laddering assay was performed to assess endo-nucleosomal fragmentation. The MTT assay results revealed that only the ethyl acetate extract has anticancer effects on human breast cancer cells (MCF-7). Half maximal Inhibitory Concentration (IC50) of the ethyl acetate extract was found to be 24.5±0.12 μg mL-1. Both inverted and fluorescence microscopic studies demonstrated that treated MCF-7 breast cancer cells using IC50 of the extract displayed a number of typical morphological changes. Appearance of membrane blebs, DNA condensation and fragmentation are significant signs of apoptosis, were observed. The above findings suggested that the ethyl acetate extract of Goniothalamus umbrosus has potential therapeutic effect towards human breast cancer cells that requires further investigations in future

Investigations of antioxidant and antibacterial activities of Typhonium flagelliforme (Lodd.) Blume leaves.

The antioxidant and antibacterial activity of different extracts from of Typhonium flagelliforme (L.) Blume leave (family: Araceae) commonly called 'Rodent Tuber' was assessed towards different antioxidant models as well as in selected bacteria. None of the extracts showed significant activity against the selected strains. The only exception is hexane extract (2.0±0.15 mm diameter) against Pseudomonas aeruginosa. The positive control, Streptomycin had shown zone of inhibition of 20±1.5, 20±1.3, 23±1.5 and 23±1.0 mm in Methicillin Resistant Staphylococcus aureus, Pseudomonas aeruginosa, Salmonella choleraesuis and Bacillus subtilis, respectively. All the extracts were subjected to screening for their possible antioxidant activity. Two complementary test systems, namely DPPH free radical scavenging and total phenolic compounds, were used for the analysis. The results showed that the inhibitory activity of Dichloromethane (60.7±3.2%) and Methanol (60.1±2.3%) extracts were comparatively commendable inhibition capacity when compared to the positive control BHT (95.3±1.3%). The total phenolic content of Methanol extracts (5.69±0.15 GAE mg g-1 extract) was superior to all other extracts, followed by dichloromethane and ethyl acetate. Considering all the results collectively T. flagelliforme appears to be a promising plant demonstrating antioxidant activity that requires further investigation

Chemical composition, antioxidant and antibacterial properties of the essentail oils of Etlingera elatior and Cinnamomum pubescens Kochummen

BACKGROUND: Plant essential oils are widely used as fragrances and flavours. Therefore, the essential oils from the leaves of Cinnamomum pubescens Kochummen (CP) and the whole plant of Etlingera elatior (EE) were investigated for their antioxidant, antibacterial and phytochemical properties. RESULTS: CP and EE were found to contain appreciable levels of total phenolic contents (50.6 and 33.41 g kg(-1) as gallic acid equivalent) and total flavonoid contents (205.6 and 244.8 g kg(-1) as rutin equivalent), respectively. DPPH free radical scavenging activity of CP is superior to EE (P < 0.05) showing IC(50) of 77.2 and 995.1 µg mL(-1), respectively. Methicillin-resistant Staphylococcus aureus (MRSA), Bacillus subtilis, Pseudomonas aeruginosa and Salmonella choleraesuis were tested against CP and EE. Only MRSA was the most susceptible bacteria to CP. GC/MS studies resulted in the identification of 79 and 73 compounds in CP and EE, respectively. The most abundant components of EE included β-pinene (24.92%) and 1-dodecene (24.31%). While the major compound in CP were 1,6-octadien-3-ol,3,7-dimethyl (11.55%), cinnamaldehyde (56.15%) and 1-phenyl-propane-2,2-diol diethanoate (11.38%). CONCLUSION: This study suggests that the essential oils from Cinnamomum pubescens Kochummen and Etlingera elatior could be potentially used as a new source of natural antioxidant and antibacterial in the food and pharmaceutical industries

Apoptosis effect of girinimbine isolated from Murraya koenigii on lung cancer cells in vitro

Murraya koenigii Spreng has been traditionally claimed as a remedy for cancer. The current study investigated the anticancer effects of girinimbine, a carbazole alkaloid isolated from Murraya koenigii Spreng, on A549 lung cancer cells in relation to apoptotic mechanistic pathway. Girinimbine was isolated from Murraya koenigii Spreng. The antiproliferative activity was assayed using MTT and the apoptosis detection was done by annexin V and lysosomal stability assays. Multiparameter cytotoxicity assays were performed to investigate the change in mitochondrial membrane potential and cytochrome c translocation. ROS, caspase, and human apoptosis proteome profiler assays were done to investigate the apoptotic mechanism of cell death. The MTT assay revealed that the girinimbine induces cell death with an IC50 of 19.01 μM. A significant induction of early phase of apoptosis was shown by annexin V and lysosomal stability assays. After 24 h treatment with 19.01 μM of girinimbine, decrease in the nuclear area and increase in mitochondrial membrane potential and plasma membrane permeability were readily visible. Moreover the translocation of cytochrome c also was observed. Girinimbine mediates its antiproliferative and apoptotic effects through up- and downregulation of apoptotic and antiapoptotic proteins. There was a significant involvement of both intrinsic and extrinsic pathways. Moreover, the upregulation of p53 as well as the cell proliferation repressor proteins, p27 and p21, and the significant role of insulin/IGF-1 signaling were also identified. Moreover the caspases 3 and 8 were found to be significantly activated. Our results taken together indicated that girinimbine may be a potential agent for anticancer drug development

In vivo and in vitro genotoxic effects of zerumbone.

Zerumbone (ZER) is derived from Zingiber zerumbet smith from the Zingiberaceae family. It has been shown to have anti-cancer and apoptosis-inducing properties against various human tumour cells. The aim of our study was to assess the genotoxic effects of ZER in cultured human peripheral blood lymphocytes, Chinese Hamster Ovary (CHO) cells and rat bone marrow polychromatic erythrocytes (PCEs) using micronucleus test (MN). All in vitro treatments were carried out in the absence of any exogenous metabolic activation system. Mitomycin C (MMC) was used as a positive control for in vitro treatments, while cisplatin was used as a positive micronucleus inducer in rat bone marrow PCEs. ZER at high concentrations induced an apparent signifi cant increase in the frequency of micronuclei in vivo (1000 mg/kg b.w) and in vitro (40 and 80 μM) compared to concurrent control values. Our in vivo and in vitro cytogenotoxicity studies suggest that high doses of ZER may be genotoxic and cytotoxic

Energy-dispersive x-ray microanalysis of elements' content of medicinal plants used traditionally as anticancer cure

This present study aims to investigate, the elemental analysis of some medicinal plants used traditionally as anticancer cure by local Malaysians. Element's content of these plants was carried out using Energy Dispersive X-ray Microanalysis combined with Variable Pressure Scanning Electron Microscope microanalysis (EDX). In this study, elements' contents from the leaves of Goniothalamus umbrosus, Kaempferia galangal, Gynura procumbens, Morinda citrifollia, Lawsonia inermis and Baringtonia racemosa using EDX technique. The elemental distribution revealed the presence of C, O, Mg, P, S, Cl, K, Ca, Al, Si and Fe, in anti-cancer medicinal plants and their relative weight percentages were estimated. These elements may be responsible for the biomedicinal properties of these plants, which based on anticancer properties. This supports the traditional usage of these plants as anti-tumor