Pentacyclic and hexacyclic cucurbitacins from Elaeocarpuspetiolatus.

Four undescribed cucurbitacins, designated as petiolaticins A-D, and four known cucurbitacins were isolated from the bark and leaves of Elaeocarpus petiolatus (Jack) Wall. Their chemical structures were elucidated based on detailed analyses of the NMR and MS data. The absolute configuration of petiolaticin A was also determined by X-ray diffraction analysis. Petiolaticin A represents a cucurbitacin derivative incorporating a 3,4-epoxyfuranyl-bearing side chain, while petiolaticin B possesses a furopyranyl unit fused to the tetracyclic cucurbitane core structure. Petiolaticins A, B, and D were evaluated in vitro against a panel of human breast, pancreatic, and colorectal cancer cell lines. Petiolaticin A exhibited the greatest cytotoxicity against the MDA-MB-468, MDA-MB-231, MCF-7, and SW48 cell lines (IC50 7.4, 9.2, 9.3, and 4.6 µM, respectively). Additionally, petiolaticin D, 16α,23α-epoxy-3ß,20ß-dihydroxy-10αH,23ßH-cucurbit-5,24-dien-11-one, and 16α,23α-epoxy-3ß,20ß-dihydroxy-10αH,23ßH-cucurbit-5,24-dien-11-one 3-O-ß-D-glucopyranoside were tested for their ability to inhibit cell entry of a pseudotyped virus bearing the hemagglutinin envelope protein of a highly pathogenic avian influenza virus. Petiolaticin D showed the highest inhibition (44.3%), followed by 16α,23α-epoxy-3ß,20ß-dihydroxy-10αH,23ßH-cucurbit-5,24-dien-11-one (21.0%), and 16α,23α-epoxy-3ß,20ß-dihydroxy-10αH,23ßH-cucurbit-5,24-dien-11-one 3-O-ß-D-glucopyranoside showed limited inhibition (9.0%). These preliminary biological assays have demonstrated that petiolaticins A and D possess anticancer and antiviral properties, respectively, which warrant for further investigations.

Virucidal activity of Garcinia parvifolia leaf extracts in animal cell culture.

BACKGROUND: Garcinia species contain bioactive compounds such as flavonoids, xanthones, triterpernoids, and benzophenones with antibacterial, antifungal, anti-inflammatory, and antioxidant activities. In addition, many of these compounds show interesting biological properties such as anti-human immunodeficiency virus activity. Garcinia parvifolia is used in traditional medicine. Currently, the antiviral activity of G. parvifolia is not known. METHODS: This study was conducted to determine the effects of ethyl acetate (45 L Ea), ethanol (45 L Et), and hexane (45 L H) leaf extracts of G. parvifolia on the infectivity of pseudorabies virus (PrV) in Vero cells. The antiviral effects of the extracts were determined by cytopathic effect (CPE), inhibition, attachment, and virucidal assays. RESULTS: The 50% cytotoxicity concentration (CC50) values obtained were 237.5, 555.0, and < 1.25 µg/mL for 45 L Ea, 45 L Et, and 45 L H, respectively. The 45 L Ea showed the greatest viral inhibition potency of 75% at 125 µg/mL. Both 45 L Ea and 45 l Et caused 100% residual viral inhibition at 250 µg/mL. The selectivity index values for 45 L Ea, 45 L Et, and 45 L H were 2.65, 1.75, and 0.10 showing that 45 L Ea had the greatest antiviral activity among the three extracts. CONCLUSION: This study showed that ethyl acetate is the best solvent to be used to obtain extract from G. parvifolia leaves with potent antiviral activities.

Epitope Presentation of Dengue Viral Envelope Glycoprotein Domain III on Hepatitis B Core Protein Virus-Like Particles Produced in Nicotiana benthamiana.

Dengue fever is currently ranked as the top emerging tropical disease, driven by increased global travel, urbanization, and poor hygiene conditions as well as global warming effects which facilitate the spread of Aedes mosquitoes beyond their current distribution. Today, more than 100 countries are affected most of which are tropical Asian and Latin American nations with limited access to medical care. Hence, the development of a dengue vaccine that is dually cost-effective and able to confer a comprehensive protection is ultimately needed. In this study, a consensus sequence of the antigenic dengue viral glycoprotein domain III (cEDIII) was used aiming to provide comprehensive coverage against all four circulating dengue viral serotypes and potential clade replacement event. Utilizing hepatitis B tandem core technology, the cEDIII sequence was inserted into the immunodominant c/e1 loop region so that it could be displayed on the spike structures of assembled particles. The tandem core particles displaying cEDIII epitopes (tHBcAg-cEDIII) were successfully produced in Nicotiana benthamiana via Agrobacterium-mediated transient expression strategy to give a protein of ∼54 kDa, detected in both soluble and insoluble fractions of plant extracts. The assembled tHBcAg-cEDIII virus-like particles (VLPs) were also visualized from transmission electron microscopy. These VLPs had diameters that range from 32 to 35 nm, presenting an apparent size increment as compared to tHBcAg control particles without cEDIII display (namely tEL). Mice immunized with tHBcAg-cEDIII VLPs showed a positive seroconversion to cEDIII antigen, thereby signifying that the assembled tHBcAg-cEDIII VLPs have successfully displayed cEDIII antigen to the immune system. If it is proven to be successful, tHBcAg-cEDIII has the potential to be developed as a cost-effective vaccine candidate that confers a simultaneous protection against all four infecting dengue viral serotypes.

Synergistic Apoptotic Effects of Tocotrienol Isomers and Acalypha wilkesiana on A549 and U87MG Cancer Cells.

Recent studies suggested that combined treatment approaches can be used to improve anticancer potency and circumvent the limitations of high-dose tocotrienols administration. Acalypha wilkesiana is a medicinal plant that has been used as an adjunct treatment for cancers in traditional medicine. Herein, the effects of single and combined treatments of ß-, γ- and δ-tocotrienols and ethyl acetate extract (9EA) of Acalypha wilkesiana on lung (A549) and brain (U87MG) cancer cells were investigated. γ- and δ-tocotrienols exhibited higher potent antiproliferative effects against A549 (12.1 µg/ml and 13.6 µg/ml) and U87MG cells (3.3 µg/ml and 5.2 µg/ml) compared to ß-tocotrienols (9.4 µg/ml and 92.4 µg/ml), respectively. Whereas, 9EA induced potent antiproliferative effects against U87MG cells only (2.0 µg/ml). Combined treatments of tocotrienols and 9EA induced a synergistic growth inhibition with up to 8.4-fold reduction in potent doses of ß-, γ- and δ-tocotrienols on A549 cells. Apoptotic features were also evidenced on A549 cells receiving single and combined treatments. The synergism may greatly improve the therapeutic outcome for lung cancer.

Enhancement of apoptotic activities on brain cancer cells via the combination of γ-tocotrienol and jerantinine A.

BACKGROUND: γ-Tocotrienol, a vitamin E isomer possesses pronounced in vitro anticancer activities. However, the in vivo potency has been limited by hardly achievable therapeutic levels owing to inefficient high-dose oral delivery which leads to subsequent metabolic degradation. Jerantinine A, an Aspidosperma alkaloid, originally isolated from Tabernaemontana corymbosa, has proved to possess interesting anticancer activities. However, jerantinine A also induces toxicity to non-cancerous cells. PURPOSE: We adopted a combinatorial approach with the joint application of γ-tocotrienol and jerantinine A at lower concentrations in order to minimize toxicity towards non-cancerous cells while improving the potency on brain cancer cells. METHODS: The antiproliferative potency of individual γ-tocotrienol and jerantinine A as well as combined in low-concentration was firstly evaluated on U87MG cancer and MRC5 normal cells. Morphological changes, DNA damage patterns, cell cycle arrests and the effects of individual and combined low-concentration compounds on microtubules were then investigated. Finally, the potential roles of caspase enzymes and apoptosis-related proteins in mediating the apoptotic mechanisms were investigated using apoptosis antibody array, ELISA and Western blotting analysis. RESULTS: Combinatorial study between γ-tocotrienol at a concentration range (0-24µg/ml) and fixed IC20 concentration of jerantinine A (0.16µg/ml) induced a potent antiproliferative effect on U87MG cells and led to a reduction on the new half maximal inhibitory concentration of γ-tocotrienol (i.e.tIC50=1.29µg/ml) as compared to that of individual γ-tocotrienol (i.e. IC50=3.17µg/ml). A reduction on undesirable toxicity to MRC5 normal cells was also observed. G0/G1 cell cycle arrest was evident on U87MG cells receiving IC50 of individual γ-tocotrienol and combined low-concentration compounds (1.29µg/ml γ-tocotrienol + 0.16µg/ml jerantinine A), whereas, a profound G2/M arrest was evident on cells treated with IC50 of individual jerantinine A. Additionally, individual jerantinine A and combined compounds (except individual γ-tocotrienol) caused a disruption of microtubule networks triggering Fas- and p53-induced apoptosis mediated via the death receptor and mitochondrial pathways. CONCLUSIONS: These findings demonstrated that the combined use of lower concentrations of γ-tocotrienol and jerantinine A induced potent cytotoxic effects on U87MG cancer cells resulting in a reduction on the required individual concentrations and thereby minimizing toxicity of jerantinine A towards non-cancerous MRC5 cells as well as probably overcoming the high-dose limiting application of γ-tocotrienol. The multi-targeted mechanisms of action of the combination approach have shown a therapeutic potential against brain cancer in vitro and therefore, further in vivo investigations using a suitable animal model should be the way forward.

Sensitivity enhancement of graphene/zinc oxide nanocomposite-based electrochemical impedance genosensor for single stranded RNA detection.

An efficient electrochemical impedance genosensing platform has been constructed based on graphene/zinc oxide nanocomposite produced via a facile and green approach. Highly pristine graphene was synthesised from graphite through liquid phase sonication and then mixed with zinc acetate hexahydrate for the synthesis of graphene/zinc oxide nanocomposite by solvothermal growth. The as-synthesised graphene/zinc oxide nanocomposite was characterised with scanning electron microscopy (SEM), transmission electron microscopy (TEM), Raman spectroscopy and X-ray diffractometry (XRD) to evaluate its morphology, crystallinity, composition and purity. An amino-modified single stranded DNA oligonucleotide probe synthesised based on complementary Coconut Cadang-Cadang Viroid (CCCVd) RNA sequence, was covalently bonded onto the surface of graphene/zinc oxide nanocomposite by the bio-linker 1-pyrenebutyric acid N-hydroxysuccinimide ester. The hybridisation events were monitored by electrochemical impedance spectroscopy (EIS). Under optimised sensing conditions, the single stranded CCCVd RNA oligonucleotide target could be quantified in a wide range of 1.0×10-11M to 1.0×10-6 with good linearity (R =0.9927), high sensitivity with low detection limit of 4.3×10-12M. Differential pulse voltammetry (DPV) was also performed for the estimation of nucleic acid density on the graphene/zinc oxide nanocomposite-modified sensing platform. The current work demonstrates an important advancement towards the development of a sensitive detection assay for various diseases involving RNA agents such as CCCVd in the future.

Exceedingly Higher co-loading of Curcumin and Paclitaxel onto Polymer-functionalized Reduced Graphene Oxide for Highly Potent Synergistic Anticancer Treatment.

Metastasis of lung carcinoma to breast and vice versa accounts for one of the vast majority of cancer deaths. Synergistic treatments are proven to be the effective method to inhibit malignant cell proliferation. It is highly advantageous to use the minimum amount of a potent toxic drug, such as paclitaxel (Ptx) in ng/ml together with a natural and safe anticancer drug, curcumin (Cur) to reduce the systemic toxicity. However, both Cur and Ptx suffer from poor bioavailability. Herein, a drug delivery cargo was engineered by functionalizing reduced graphene oxide (G) with an amphiphilic polymer, PF-127 (P) by hydrophobic assembly. The drugs were loaded via pi-pi interactions, resulting in a nano-sized GP-Cur-Ptx of 140 nm. A remarkably high Cur loading of 678 wt.% was achieved, the highest thus far compared to any other Cur nanoformulations. Based on cell proliferation assay, GP-Cur-Ptx is a synergistic treatment (CI < 1) and is highly potent towards lung, A549 (IC50 = 13.24 µg/ml) and breast, MDA-MB-231 (IC50 = 1.450 µg/ml) cancer cells. These positive findings are further confirmed by increased reactive oxygen species, mitochondrial membrane potential depletion and cell apoptosis. The same dose treated on normal MRC-5 cells shows that the system is biocompatible and cancerous cell-specific.

Antiviral and virucidal activities of Duabanga grandiflora leaf extract against Pseudorabies virus in vitro.

BACKGROUND: Duabanga grandiflora or known in Malaysia as Berembang Bukit, Megawasih, or Pedada Bukit, is a native plant of the Southeast Asian countries. In this study, the anti-viral properties of D. grandiflora were investigated. METHODS: The D. grandiflora leaf extracts were obtained with ethyl acetate, hexane, and ethanol as solvents and labelled 37 leaf ethyl acetate (37 L EA), 37 leaf hexane (37 L H), 37 leaf ethanol (37 L ET), respectively. The cytotoxicity of the extracts on Vero cells were determined by the 3-(4,5-Diamethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) assay. RESULTS: Among extracts, 37 L EA was most cytotoxic to Vero cells, followed by 37 L H and 37 L ET, with CC50 of 218, 833, and >1000 µg/mL, respectively. The cytopathic effect (CPE) and plaque reduction, inhibition, and virucidal assays and the selective index (SI) were employed to determine the effect of the extracts on infectivity and replication of pseudorabies virus (PrV) in Vero cells. The D. grandiflora leaf extracts showed dose-dependent antiviral activities, with higher activities at high doses. The 37 L ET and 37 L EA showed anti-viral effects through plaque formation and viral replication inhibitions, and virucidal property. The SI of the 37 L ET and 37 L EA by the viral replication inhibition assay was 8.3 and 1.9, respectively, and by the CPE reduction assay, 6.7 and 2.9, respectively. CONCLUSION: Ethanol is the best solvent for the preparation of D. grandiflora leaf extract as an antiviral agent.

Synergistic cytotoxic effects of combined δ-tocotrienol and jerantinine B on human brain and colon cancers.

ETHNOPHARMACOLOGICAL RELEVANCE: The genus Tabernaemontana has widespread distribution throughout tropical and subtropical parts of the world, i.e. Africa, Asia and America which has long been used for treatments of different disease conditions including tumours, wounds, syphilis, stomach ache and headache. Some Tabernaemontana species are used for treatment of piles, spleen and abdominal tumours in India. In particular, the leaf of Tabernaemontana corymbosa is used for treatment of tumours in Bangladesh. Parts of the plant or whole plants are used as decoctions, steam bath, powder and ointments. AIM OF STUDY: The present study was undertaken to study the mechanism of apoptosis induction in human glioblastoma (U87MG) and colorectal adenocarcinoma (HT-29) cancer cells by a novel indole alkaloid, jerantinine B isolated from T. corymbosa, δ-tocotrienol and the combined low-dose treatments of δ-tocotrienol with IC20 dose of jerantinine B. MATERIALS AND METHODS: Cell viability, isobologram and combinational index (CI) analyses were used to determine the pharmacological interaction between combined treatments based on the IC50 values obtained. Fluorescence and histochemical staining techniques as well as comet assay were used for evaluating the morphological changes and DNA damage pattern, respectively. The effects of treatments on microtubules, caspase activity and cell death were determined using immunofluorescence technique, caspase colorimetric and neutral red uptake assays, respectively. RESULTS: Jerantinine B, δ-tocotrienol and combined low-dose treatments induced a dose-dependent growth inhibition against U87MG and HT-29 cells selectively with less toxicity acted towards the normal MRC5 cells. Synergistic growth inhibition observed with CI values of 0.85 and 0.77 for U87MG and HT-29 cells, resulting in up to 2-fold and 3.8-fold dose reduction of δ-tocotrienol and jerantinine B, respectively. U87MG and HT-29 cells exhibited morphological features of apoptosis and double stranded DNA breaks. Individual and combined treatments induced caspase 8 and 3 activities and cell death independent of caspase activation on U87MG and HT-29 cells. An increased caspase 9 activity was also evident on U87MG and HT-29 treated with combined treatments and HT-29 cells treated with jerantinine B. Jerantinine B and combined low-dose treatments with δ-tocotrienol undoubtedly disrupted the microtubule networks. CONCLUSION: The present study demonstrated the mechanism for cytotoxic potency of δ-tocotrienol and jerantinine B against U87MG and HT-29 cells. Furthermore, combined low-dose treatments induced concurrent synergistic inhibition of cancer cell growth with concomitant dose reduction thus minimizing toxicity to normal cells and improving potency of δ-tocotrienol and jerantinine B.

A review on ethnobotany, pharmacology and phytochemistry of Tabernaemontana corymbosa.

OBJECTIVES: Tabernaemontana is a genus from the plant family, Apocynaceae with vast medicinal application and widespread distribution in the tropics and subtropics of Africa, Americas and Asia. The objective of this study is to critically evaluate the ethnobotany, medicinal uses, pharmacology and phytochemistry of the species, Tabernaemontana corymbosa (Roxb. ex Wall.) and provide information on the potential future application of alkaloids isolated from different parts of the plant. KEY FINDINGS: T. corymbosa (Roxb. ex Wall.) parts are used as poultice, boiled juice, decoctions and infusions for treatment against ulceration, fracture, post-natal recovery, syphilis, fever, tumours and orchitis in Malaysia, China, Thailand and Bangladesh. Studies recorded alkaloids as the predominant phytochemicals in addition to phenols, saponins and sterols with vast bioactivities such as antimicrobial, analgesic, anthelmintic, vasorelaxation, antiviral and cytotoxicity. SUMMARY: An evaluation of scientific data and traditional medicine revealed the medicinal uses of different parts of T. corymbosa (Roxb. ex Wall.) across Asia. Future studies exploring the structure-bioactivity relationship of alkaloids such as jerantinine and vincamajicine among others could potentially improve the future application towards reversing anticancer drug resistance.

Inhibition of penicillin-binding protein 2a (PBP2a) in methicillin resistant Staphylococcus aureus (MRSA) by combination of ampicillin and a bioactive fraction from Duabanga grandiflora.

BACKGROUND: The inhibition of penicillin-binding protein 2a (PBP2a) is a promising solution in overcoming resistance of methicillin resistance Staphylococcus aureus (MRSA). A potential approach in achieving this is by combining natural product with currently available antibiotics to restore the activity as well as to amplify the therapeutic ability of the drugs. We studied inhibition effects of a bioactive fraction, F-10 (isolated from the leaves of Duabanga grandiflora) alone and in combination with a beta-lactam drug, ampicillin on MRSA growth and expression of PBP2a. Additionally, phytochemical analysis was conducted on F-10 to identify the classes of phytochemicals present. METHODS: Fractionation of the ethyl acetate leaf extract was achieved by successive column chromatography which eventually led to isolation of an active fraction, F-10. Both extract and F-10 were analyzed for the presence of major classes of phytochemicals in addition to obtaining a high performance liquid chromatography (HPLC) profile to reveal the complexity of the fraction F-10. Broth microdilution method was employed to determine minimum inhibitory concentration (MIC) of the extract and fractions against MRSA. Evaluation of synergistic activity of the active fraction with ampicillin was determined using checkerboard methodand kinetic growth experiments. Effect of combination treatments on expression of PBP2a, a protein that confers resistance to beta-lactam antibiotics, was elucidated with the Western blot assay. RESULTS: MIC of F-10 against MRSA was 750 mg/L which showed an improved activity by 4-fold compared to its crude extract (MIC = 3000 mg/L). Phytochemical analysis revealed occurrence of tannins, saponin, flavonoids, sterols, and glycosides in F10 fraction. In FIC index interpretation, the most synergistic activity was achieved for combinations of 1/64 × MIC ampicillin + 1/4 × MIC F-10. The combination also evidently inhibited MRSA growth in kinetic growth curve assay. As a result of this synergistic interaction, MIC of ampicillin against MRSA was reduced to 0.78 mg/L (64-fold) from initial value of 50 mg/L. Western blot analysis suggested inhibition of PBP2a in MRSA cultures grown in synergistic combination treatment in which no PBP2a band was expressed. CONCLUSIONS: The results demonstrated synergism between fraction F-10 of D. grandiflora with ampicillin in suppressing MRSA growth via PBP2a inhibition.

Prevention of cell-surface attachment and reduction of penicillin-binding protein 2a (PBP2a) level in methicillin-resistant Staphylococcus aureus biofilms by Acalypha wilkesiana.

BACKGROUND: Formation of biofilm is known to enhance the virulence of methicillin-resistance Staphylococcus aureus (MRSA), which is associated with persistent infections in hospital settings. The biofilm layer essentially forms a protective barrier encapsulating the bacterial colony and thus reduces the effectiveness of chemotherapeutics. We have isolated 9EA-FC-B bioactive fraction from Acalypha wilkesiana Müll. Arg. that reverses ampicillin resistant in MRSA through inhibition of the antibiotic resistant protein, penicillin-binding protein 2a (PBP2a). In this study, we aimed to investigate the effects of 9EA-FC-B on MRSA biofilm forming capacity. METHODS: Inhibition of biofilm production and microtiter attachment assays were employed to study the anti-biofilm activity of 9EA-FC-B, while latex agglutination test was performed to investigate the effect on PBP2a in the biofilm matrix. We also attempted to characterise the chemical components of the fraction using high performance liquid chromatography (HPLC) and phytochemical analysis. RESULTS: Fraction 9EA-FC-B and ampicillin exhibited similar inhibitory effect on MRSA's biofilm production at their respective minimum inhibitory concentrations (81.56% vs 84.49%, respectively). However, the test fraction was more effective in suppressing cell surface attachment (90.85%) compared to ampicillin (37.8%). Interestingly, ampicillin enhanced the level PBP2a and in the contrary 9EA-FC-B attenuated the production of the resistant protein in the bioflim matrix. HPLC and phytochemical analysis revealed that 9EA-FC-B fraction is a complex mixture containing tannins, saponins, sterol/steroids, and glycosides. CONCLUSIONS: Bioactive fraction 9EA-FC-B inhibited the production of MRSA biofilm by preventing the initial cell-surface attachment and reducing the amount PBP2a in the matrix. PBP2a found in the biofilm matrix is believed to have a role in the development of virulence in MRSA.

Inhibitory effect of Duabanga grandiflora on MRSA biofilm formation via prevention of cell-surface attachment and PBP2a production.

Formation of biofilms is a major factor for nosocomial infections associated with methicillin-resistance Staphylococcus aureus (MRSA). This study was carried out to determine the ability of a fraction, F-10, derived from the plant Duabanga grandiflora to inhibit MRSA biofilm formation. Inhibition of biofilm production and microtiter attachment assays were employed to study the anti-biofilm activity of F-10, while latex agglutination test was performed to study the influence of F-10 on penicillin-binding protein 2a (PBP2a) level in MRSA biofilm. PBP2a is a protein that confers resistance to beta-lactam antibiotics. The results showed that, F-10 at minimum inhibitory concentration (MIC, 0.75 mg/mL) inhibited biofilm production by 66.10%; inhibited cell-surface attachment by more than 95%; and a reduced PBP2a level in the MRSA biofilm was observed. Although ampicilin was more effective in inhibiting biofilm production (MIC of 0.05 mg/mL, 84.49%) compared to F-10, the antibiotic was less effective in preventing cell-surface attachment. A higher level of PBP2a was detected in ampicillin-treated MRSA showing the development of further resistance in these colonies. This study has shown that F-10 possesses anti-biofilm activity, which can be attributed to its ability to reduce cell-surface attachment and attenuate the level of PBP2a that we postulated to play a crucial role in mediating biofilm formation.

Alkaloid extracts of Ficus species and palm oil-derived tocotrienols synergistically inhibit proliferation of human cancer cells.

Tocotrienols have been reported to possess anticancer effects other than anti-inflammatory and antioxidant activities. This study explored the potential synergism of antiproliferative effects induced by individual alkaloid extracts of Ficus fistulosa, Ficus hispida and Ficus schwarzii combined with δ- and γ-tocotrienols against human brain glioblastoma (U87MG), lung adenocarcinoma (A549) and colorectal adenocarcinoma (HT-29) cells. Cell viability and morphological results demonstrated that extracts containing a mixture of alkaloids from the leaves and bark of F. schwarzii inhibited the proliferation of HT-29 cells, whereas the alkaloid extracts of F. fistulosa inhibited the proliferation of both U87MG and HT-29 cells and showed synergism in combined treatments with either δ- or γ-tocotrienol resulting in 2.2-34.7 fold of reduction in IC50 values of tocotrienols. The observed apoptotic cell characteristics in conjunction with the synergistic antiproliferative effects of Ficus species-derived alkaloids and tocotrienols assuredly warrant future investigations towards the development of a value-added chemotherapeutic regimen against cancers.

Cytotoxicity and apoptotic activities of alpha-, gamma- and delta-tocotrienol isomers on human cancer cells.

BACKGROUND: Tocotrienols, especially the gamma isomer was discovered to possess cytotoxic effects associated with the induction of apoptosis in numerous cancers. Individual tocotrienol isomers are believed to induce dissimilar apoptotic mechanisms in different cancer types. This study was aimed to compare the cytotoxic potency of alpha-, gamma- and delta-tocotrienols, and to explore their resultant apoptotic mechanisms in human lung adenocarcinoma A549 and glioblastoma U87MG cells which are scarcely researched. METHODS: The cytotoxic effects of alpha-, gamma- and delta-tocotrienols in both A549 and U87MG cancer cells were first determined at the cell viability and morphological aspects. DNA damage types were then identified by comet assay and flow cytometric study was carried out to support the incidence of apoptosis. The involvements of caspase-8, Bid, Bax and mitochondrial membrane permeability (MMP) in the execution of apoptosis were further expounded. RESULTS: All tocotrienols inhibited the growth of A549 and U87MG cancer cells in a concentration- and time-dependent manner. These treated cancer cells demonstrated some hallmarks of apoptotic morphologies, apoptosis was further confirmed by cell accumulation at the pre-G1 stage. All tocotrienols induced only double strand DNA breaks (DSBs) and no single strand DNA breaks (SSBs) in both treated cancer cells. Activation of caspase-8 leading to increased levels of Bid and Bax as well as cytochrome c release attributed by the disruption of mitochondrial membrane permeability in both A549 and U87MG cells were evident. CONCLUSIONS: This study has shown that delta-tocotrienol, in all experimental approaches, possessed a higher efficacy (shorter induction period) and effectiveness (higher induction rate) in the execution of apoptosis in both A549 and U87MG cancer cells as compared to alpha- and gamma-tocotrienols. Tocotrienols in particular the delta isomer can be an alternative chemotherapeutic agent for treating lung and brain cancers.

Reversal of ampicillin resistance in MRSA via inhibition of penicillin-binding protein 2a by Acalypha wilkesiana.

The inhibitory activity of a semipure fraction from the plant, Acalypha wilkesiana assigned as 9EA-FC-B, alone and in combination with ampicillin, was studied against methicillin-resistant Staphylococcus aureus (MRSA). In addition, effects of the combination treatment on PBP2a expression were investigated. Microdilution assay was used to determine the minimal inhibitory concentrations (MIC). Synergistic effects of 9EA-FC-B with ampicillin were determined using the fractional inhibitory concentration (FIC) index and kinetic growth curve assay. Western blot experiments were carried out to study the PBP2a expression in treated MRSA cultures. The results showed a synergistic effect between ampicillin and 9EA-FC-B treatment with the lowest FIC index of 0.19 (synergism ≤ 0.5). The presence of 9EA-FC-B reduced the MIC of ampicillin from 50 to 1.56 µg mL(-1). When ampicillin and 9EA-FC-B were combined at subinhibitory level, the kinetic growth curves were suppressed. The antibacterial effect of 9EA-FC-B and ampicillin was shown to be synergistic. The synergism is due the ability of 9EA-FC-B to suppress the activity of PBP2a, thus restoring the susceptibility of MRSA to ampicillin. Corilagin was postulated to be the constituent responsible for the synergistic activity showed by 9EA-FC-B.

Search for antibacterial agents from Malaysian rainforest and tropical plants.

The world's rainforests hold untold potential for drug discovery. Rainforest plants are thought to contain evolved defensive active metabolites of greater diversity compared to plants from temperate regions. In recent years, the interest and overall output from pharmaceutical companies on novel antibacterial agents has diminished at a time when there is a critical need for them to fight the threat of resistance. In this study, we have investigated the antimicrobial properties of 21 flowering plants from 16 different families against six bacterial strains consisting of two Gram negative and four Gram positive. Using the pour plate disc diffusion technique, almost all extracts from these plants were found to be active against some of the bacterial strains tested. The most interesting and active plants with broad spectrum activities include Duabanga grandiflora, Acalypha wilkesiana and Pseuduvaria macrophylla where the minimum inhibitory concentration, minimum bactericidal concentration and phytochemical analysis were carried out. This is the first report describing the antimicrobial and phytochemical properties of D. grandiflora and P. macrophylla. Our findings support the utilisation of higher plant species in the search for new antimicrobial molecules to combat new emerging infective diseases and the problem of drug resistant pathogens.

Optimal methods for evaluating antimicrobial activities from plant extracts.

The search for antimicrobial agents from plants has been a growing interest in the last few decades. However, results generated from many of these studies cannot be directly compared due to the absence of standardization in particular antimicrobial methods employed. The need for established methods with consistent results for the evaluation of antimicrobial activities from plant extracts has been proposed by many researchers. Nevertheless, there are still many studies reported in the literature describing different methodologies. The aim of this study was to find optimal methods to give consistent quantitative antimicrobial results for studying plant extracts. Three different agar-based assays (pour plate disc diffusion (PPDD), streak plate disc diffusion (SPDD) and well-in agar (WA)) and one broth-based (turbidometric (TB)) assay were used in this study. Extracts from two plant species (Duabanga grandiflora and Acalypha wilkesiana) were tested on two bacterial species, namely Escherichia coli and Staphylococcus aureus. Amongst the agar-based assays, PPDD produced the most reproducible results. TB was able to show the inhibitory effects of the test samples on the growth kinetic of the bacteria including plant extracts with low polarity. We propose that both agar- (i.e PPDD) and broth-based assays should be employed when assessing the antimicrobial activity of plant crude extracts.