Three Australian Lepidosperma Labill. Species as sources of prenylated and oxyprenylated derivatives of piceatannol, resveratrol and pinosylvin: Melatoninergic binding and inhibition of quinone reductase 2.

Prenylated and hydroxyprenylated piceatannol, resveratrol and pinosylvin derivatives were isolated from resin produced by three Australian Lepidosperma Labill. Species (Cyperaceae). From L. congestum R.Br. one known compound, 3',5'-bis-prenyl-E-resveratrol, and five undescribed compounds were isolated, 3'-O-prenyl-5'-prenyl-E-piceatannol, 5',6'-bis-prenyl-E-piceatannol, 5'-prenyl-E-piceatannol, 3',5'-bis(3-hydroxy-3-methylbutyl)-E-resveratrol and 3',5'-bis-E-hydroxyprenyl-E-resveratrol. From L. gunnii Boeckeler one undescribed compound was isolated, 3'-E-hydroxyprenyl-5'-Z-hydroxyprenyl-E-resveratrol. From L. laterale R.Br. six undescribed compounds were isolated, 3-O-prenyl-E-pinosylvin, 3-O-Z-hydroxyprenyl-E-pinosylvin, 3'-Z-hydroxyprenyl-E-resveratrol, 3-O-Z-hydroxyprenyl-E-resveratrol, 3-O-Z-hydroxyprenyl-4'-O-methyl-E-resveratrol, and 3-O-prenyl-3'-δ,δ'-dihydroxyprenyl-E-resveratrol. Compounds, including a reference compound 3-O-prenyl-3'-O-methyl-E-piceatannol, were screened in an assay for melatoninergic binding to MT1 and MT2 receptors and binding to QR2/MT3 enzyme, and for inhibition of QR2/MT3 in a functional assay. Strong binding was observed for 3-O-Z-hydroxyprenyl-E-resveratrol with a Ki of 0.022 nM and the strongest inhibition of QR2/MT3 observed was for the reference compound, 3-O-prenyl-3'-O-methyl-E-piceatannol, with an inhibition of 61% at 1 µM and 95% at 10 µM. The three most active binders and inhibitors of QR2/MT3 were found to have a common substructure corresponding to 3-O-prenylresveratrol.

Kangaroo Island propolis types originating from two Lepidosperma species and Dodonaea humilis.

The endemic Australian plants Lepidosperma sp. Flinders Chase (Cyperaceae), Lepidosperma viscidum (Cyperaceae) and Dodonaea humilis (Sapindaceae) were found to be the botanical origin of three propolis types found on Kangaroo Island identified by TLC and 1H NMR matching of propolis and plant resin analytical profiles. Resin samples extracted from the plant, Lepidosperma sp. Flinders Chase, were chromatographically fractionated to give: methyl 3-phenyl-2-(E-cinnamoyloxy)propanoate (1), 3-(E-8-methoxy-8-oxo-3,7-dimethyloct-2-enyl)-4-hydroxy-E-cinnamic acid (2), 3-(E-6,7-dihydroxy-3,7-dimethyloct-2-enyl)-4-hydroxy-E-cinnamic acid (3), previously undescribed; and the known stilbenes, 2-prenyl-3,5-dihydroxy-E-stilbene (6) and 2-prenyl-3-methoxy-5-hydroxy-E-stilbene (7). The resin from L. viscidum gave: 5'-(E-4-hydroxy-3-methylbut-2-enyl)-4,2',4'-trihydroxydihydrochalcone (4); 5'-(E-4-hydroxy-3-methylbut-2-enyl)-4'-methoxy-4,2'-dihydroxydihydrochalcone (5), previously undescribed; and three known flavanones, farrerol (8), 5,7,3',5'-tetrahydroxy-6,8-dimethylflavanone (9) and 5,7,3',5'-tetrahydroxy-6-methylflavanone (10). The major constituent in the propolis identified as being sourced from D. humilis was identified as 6,8-diprenyl-5,7,3',4'-tetrahydroxyflavanone (11), a known compound identified in several unrelated plant species.

A sedge plant as the source of Kangaroo Island propolis rich in prenylated p-coumarate ester and stilbenes.

Propolis samples from Kangaroo Island, South Australia, were investigated for chemical constituents using high-field nuclear magnetic resonance spectral profiling. A type of propolis was found containing a high proportion of prenylated hydroxystilbenes. Subsequently, the botanical origin of this type of propolis was identified using a beehive propolis depletion method and analysis of flora. Ligurian honey bees, Apis mellifera ligustica Spinola, were found to produce propolis from resin exuded by the Australian native sedge plant Lepidosperma sp. Montebello (Cyperaceae). The plants, commonly known as sword sedge, were found to have resin that matched with the propolis samples identified as the most abundant propolis type on the island containing C- and O-prenylated tetrahydroxystilbenes (pTHOS) in addition to a small amount of prenylated p-coumarate. The isolation of five pTHOS not previously characterized are reported: (E)-4-(3-methyl-2-buten-1-yl)-3,4',5-trihydroxy-3'-methoxystilbene, (E)-2,4-bis(3-methyl-2-buten-1-yl)-3,3',4',5-tetrahydroxystilbene, (E)-2-(3-methyl-2-buten-1-yl)-3-(3-methyl-2-butenyloxy)-3',4',5-trihydroxystilbene, (E)-2,6-bis(3-methyl-2-buten-1-yl)-3,3',5,5'-tetrahydroxystilbene and (E)-2,6-bis(3-methyl-2-buten-1-yl)-3,4',5-trihydroxy-3'-methoxystilbene. A National Cancer Institute 60 human cell line anticancer screen of three of these compounds showed growth inhibitory activity. The large Australasian genus Lepidosperma is identified as a valuable resource for the isolation of substances with medicinal potential.

Murine pharmacokinetics of rifapentine delivered as an inhalable dry powder.

A novel inhalable rifapentine dry powder formulation could improve pulmonary rifapentine concentrations resulting in a significantly shorter time to treat tuberculosis infection. The pharmacokinetics of rifapentine (20mg/kg) in healthy mice was compared following intratracheal (IT) and intraperitoneal (IP) administration. Plasma, bronchoalveolar lavage (BAL) and tissue samples were collected and drug levels were quantified at time points up to 24h. Concentration-time data were analysed using a mixed-effects modelling approach to provide model-based estimates of area under the concentration-time curve from time 0 to infinity (AUC0-∞). IT delivery had considerably higher peak rifapentine lung and BAL concentrations and associated AUC0-∞ compared with IP delivery. The plasma AUC0-∞ following IT dry powder delivery was ca. four-fold smaller than the value for IP delivery. Inhaled delivery of rifapentine has the potential to selectively enhance therapeutic efficacy at the pulmonary site of infection whilst minimising systemic exposure and related toxicity.

A novel inhalable form of rifapentine.

Recent murine studies found that rifapentine, dosed daily, at least halved tuberculosis treatment times compared with standard rifampicin and isoniazid-containing regimens. However, in humans, an inhalable form of rifapentine may be necessary to considerably shorten treatment duration because of the physiological barriers associated with oral therapy. The current study compares two inhalable rifapentine dry powders-a novel pure crystalline form and an amorphous form-by a series of in vitro tests. The crystalline and amorphous powders had a mass median aerodynamic size of 1.68 ± 0.03 and 1.92 ± 0.01 µm, respectively, associated with a fine particle fraction of 83.2 ± 1.2% and 68.8 ± 2.1%, respectively. A quinone degradation product was identified in the amorphous powder stored for 1 month, whereas the crystalline form remained chemically stable after storage at both 0% and 60% relative humidity, 25°C, for at least 3 months. Solubilized rifapentine was well tolerated by pulmonary tissue and macrophage cells up to approximately 50 µM. The accumulation of rifapentine within alveolar macrophage cells was significantly higher than for rifampicin, indicating enhanced delivery to infected macrophages. The novel inhalable crystalline form of rifapentine is suitable for targeted treatment of tuberculosis infection and may radically shorten treatment duration.

Preventative effect of Zingiber officinale on insulin resistance in a high-fat high-carbohydrate diet-fed rat model and its mechanism of action.

Insulin resistance is a core component of metabolic syndrome and usually precedes the development of type 2 diabetes mellitus. We have examined the preventative effect of an ethanol extract of ginger (Zingiber officinale, Zingiberaceae) on insulin resistance in a high-fat high-carbohydrate (HFHC) diet-fed rat model of metabolic syndrome. The HFHC control rats displayed severe insulin resistance, whilst rats treated with ginger extract (200 mg/kg) during HFHC diet feeding showed a significant improvement of insulin sensitivity using the homeostatic model assessment of insulin resistance (HOMA-IR) after 10 weeks (p < 0.01). An in vitro mechanistic study showed that (S)-[6]-gingerol, the major pungent phenolic principle in ginger, dose-dependently (from 50 to 150 µM) increased AMPK α-subunit phosphorylation in L6 skeletal muscle cells. This was accompanied by a time-dependent marked increment of PGC-1α mRNA expression and mitochondrial content in L6 skeletal muscle cells. These results suggest that the protection from HFHC diet-induced insulin resistance by ginger is likely associated with the increased capacity of energy metabolism by its major active component (S)-[6]-gingerol.

A rifapentine-containing inhaled triple antibiotic formulation for rapid treatment of tubercular infection.

PURPOSE: The potential for rifapentine-containing oral therapeutic regimens to significantly shorten the current six-month anti-tubercular treatment regimen is confounded by high plasma protein binding of rifapentine. Inhaled aerosol delivery of rifapentine, a more potent anti-tubercular antibiotic drug, in combination with other first-line antibiotics may overcome this limitation to deliver a high drug dose at the pulmonary site of infection. METHODS: A formulation consisting of rifapentine, moxifloxacin and pyrazinamide, with and without leucine, was prepared by spray-drying. This formulation was assessed for its physico-chemical properties, in vitro aerosol performance and antimicrobial activity. RESULTS: The antibiotic powders, with and without leucine, had similar median aerodynamic diameters of 2.58 ± 0.08 µm and 2.51 ± 0.06 µm, with a relatively high fine particle fraction of 55.5 ± 1.9% and 63.6 ± 2.0%, respectively. Although the powders were mostly amorphous, some crystalline peaks associated with the δ polymorph for the spray-dried crystalline pyrazinamide were identified. CONCLUSIONS: Stabilisation of the powder with 10% w/w leucine and protection from moisture ingress was found to be necessary to prevent overt crystallisation of pyrazinamide after long-term storage. In vitro biological assays indicated antimicrobial activity was retained after spray-drying. Murine pharmacokinetic studies are currently underway.

Identification of a Calcium Signalling Pathway of S-[6]-Gingerol in HuH-7 Cells.

Calcium signals in hepatocytes control cell growth, proliferation, and death. Members of the transient receptor potential (TRP) cation channel superfamily are candidate calcium influx channels. NF κ B activation strictly depends on calcium influx and often induces antiapoptotic genes favouring cell survival. Previously, we reported that S-[6]-gingerol is an efficacious agonist of the transient receptor potential cation channel subfamily V member 1 (TRPV1) in neurones. In this study, we tested the effect of S-[6]-gingerol on HuH-7 cells using the Fluo-4 calcium assay, RT-qPCR, transient cell transfection, and luciferase measurements. We found that S-[6]-gingerol induced a transient rise in [Ca(2+)] i in HuH-7 cells. The increase in [Ca(2+)] i induced by S-[6]-gingerol was abolished by preincubation with EGTA and was also inhibited by the TRPV1 channel antagonist capsazepine. Expression of TRPV1 in HuH-7 cells was confirmed by mRNA analysis as well as a test for increase of [Ca(2+)] i by TRPV1 agonist capsaicin and its inhibition by capsazepine. We found that S-[6]-gingerol induced rapid NF κ B activation through TRPV1 in HuH-7 cells. Furthermore, S-[6]-gingerol-induced NF κ B activation was dependent on the calcium gradient and TRPV1. The rapid NF κ B activation by S-[6]-gingerol was associated with an increase in mRNA levels of NF κ B-target genes: cIAP-2, XIAP, and Bcl-2 that encode antiapoptotic proteins.

Attenuation of Proinflammatory Responses by S-[6]-Gingerol via Inhibition of ROS/NF-Kappa B/COX2 Activation in HuH7 Cells.

Introduction. Hepatic inflammation underlies the pathogenesis of chronic diseases such as insulin resistance and type 2 diabetes mellitus. S-[6]-Gingerol has been shown to have anti-inflammatory properties. Important inflammatory mediators of interleukins include nuclear factor κ B (NF κ B) and cyclooxygenase 2 (COX2). We now explore the mechanism of anti-inflammatory effects of S-[6]-gingerol in liver cells. Methods. HuH7 cells were stimulated with IL1ß to establish an in vitro hepatic inflammatory model. Results. S-[6]-Gingerol attenuated IL1ß-induced inflammation and oxidative stress in HuH7 cells, as evidenced by decreasing mRNA levels of inflammatory factor IL6, IL8, and SAA1, suppression of ROS generation, and increasing mRNA levels of DHCR24. In addition, S-[6]-gingerol reduced IL1ß-induced COX2 upregulation as well as NF κ B activity. Similar to the protective effects of S-[6]-gingerol, both NS-398 (a selective COX2 inhibitor) and PDTC (a selective NF κ B inhibitor) suppressed mRNA levels of IL6, IL8, and SAA1. Importantly, PDTC attenuated IL1ß-induced overexpression of COX2. Of particular note, the protective effect of S-[6]-gingerol against the IL1ß-induced inflammatory response was similar to that of BHT, an ROS scavenger. Conclusions. The findings of this study demonstrate that S-[6]-gingerol protects HuH7 cells against IL1ß-induced inflammatory insults through inhibition of the ROS/NF κ B/COX2 pathway.

(S)-[6]-Gingerol inhibits TGF-ß-stimulated biglycan synthesis but not glycosaminoglycan hyperelongation in human vascular smooth muscle cells.

OBJECTIVES: (S)-[6]-Gingerol is under investigation for a variety of therapeutic uses. Transforming growth factor (TGF)-ß stimulates proteoglycan synthesis, leading to increased binding of low-density lipoproteins, which is the initiating step in atherosclerosis. We evaluated the effects of (S)-[6]-gingerol on these TGF-ß-mediated proteoglycan changes to explore its potential as an anti-atherosclerotic agent. METHODS: Purified (S)-[6]-gingerol was assessed for its effects on proteoglycan synthesis by [(35) S]-sulfate incorporation into glycosaminoglycan chains and [(35) S]-Met/Cys incorporation into proteoglycans and total proteins in human vascular smooth muscle cells. Biglycan level was assessed by real-time quantitative polymerase chain reactions and the effects of (S)-[6]-gingerol on TGF-ß signalling by assessment of the phosphorylation of Smads and Akt by western blotting. KEY FINDINGS: (S)-[6]-Gingerol concentration-dependently inhibited TGF-ß-stimulated proteoglycan core protein synthesis, and this was not secondary to inhibition of total protein synthesis. (S)-[6]-Gingerol inhibited biglycan mRNA expression. (S)-[6]-Gingerol did not inhibit TGF-ß-stimulated glycosaminoglycan hyperelongation or phosphorylation of Smad 2, in either the carboxy terminal or linker region, or Akt phosphorylation. CONCLUSIONS: The activity of (S)-[6]-gingerol to inhibit TGF-ß-stimulated biglycan synthesis suggests a potential role for ginger in the prevention of atherosclerosis or other lipid-binding diseases. The signalling studies indicate a novel site of action of (S)-[6]-gingerol in inhibiting TGF-ß responses.

Synthesis of C- and O-prenylated tetrahydroxystilbenes and O-prenylated cinnamates and their action towards cancer cells.

Synthesis of the naturally occurred C- and O-prenylated tetrahydroxystilbenes and O-prenylated cinnamates was carried out by decarbonylative Heck reaction and selenium dioxide catalysed oxidation, respectively. In the decarbonylative Heck synthetic route, fusion of benzoyl chloride and styrene derivatives was catalysed by an N-heterocyclic carbene system generated in situ by palladium acetate and 1,3-bis(2,6-diisopropylphenyl)imidazolinium chloride to form a E-tetrahydroxystilbene derivative. Formation of allyl ether was subsequently carried out by reaction of the deprotected OH in the A phenyl ring of the stilbene with 3,3-dimethylallyl bromide and a base (sodium hydride) to form O-prenylated tetrahydroxystilbene derivatives. [1,5]-Rearrangement of the isoprenyl unit from O- to C-position in the A ring was carried out at elevated temperature in the presence of magnesium silicate (Florisil) to form the corresponding C-prenylated tetrahydroxystilbene. Formation of O-prenylated cinnamate was first carried out by base catalysed allyl ether formation between 3,3-dimethylallyl bromide and hydroxycinnamic acid methyl ester. The methyl group of the isoprenyl unit was subsequently oxidized using selenium dioxide to form a terminal hydroxyl group. The prenylated tetrahydroxystilbenes and cinnamate synthesized in this study were novel derivatives of piceatannol and methyl 4-(3'-methylbut-2'-enyloxy)cinnamate isolated from propolis in Kangaroo Island, South Australia. The synthetic compounds were tested against K562 cancer cells and potent growth inhibitory activity was observed for E-1-[5-hydroxy-3-methoxy-2-(3-methyl-2-butenyl)phenyl]-2-[4-hydroxy-3-methoxyphenyl]ethene, IC50 = 0.10 µM.

Preventive and Protective Properties of Zingiber officinale (Ginger) in Diabetes Mellitus, Diabetic Complications, and Associated Lipid and Other Metabolic Disorders: A Brief Review.

Zingiber officinale (ginger) has been used as herbal medicine to treat various ailments worldwide since antiquity. Recent evidence revealed the potential of ginger for treatment of diabetes mellitus. Data from in vitro, in vivo, and clinical trials has demonstrated the antihyperglycaemic effect of ginger. The mechanisms underlying these actions are associated with insulin release and action, and improved carbohydrate and lipid metabolism. The most active ingredients in ginger are the pungent principles, gingerols, and shogaol. Ginger has shown prominent protective effects on diabetic liver, kidney, eye, and neural system complications. The pharmacokinetics, bioavailability, and the safety issues of ginger are also discussed in this update.

Propolis with high flavonoid content collected by honey bees from Acacia paradoxa.

Honey bees, Apis mellifera var ligustica, on Kangaroo Island, Australia, were found to collect propolis from the sticky exudate on the stem shoots and seed pods of an Australian endemic plant, Acacia paradoxa. Extracts of the plant stem shoots and seed pods, the propolis carried on the legs of bees and freshly collected propolis in hives contained major flavonoid components consisting of 2',3',4'-trimethoxychalcone, 2'-hydroxy-3',4'-dimethoxychalcone, 2',4'-dihydroxy-3'-methoxychalcone, 5,7-dihydroxy-2,3-dihydroflavonol 3-acetate (pinobanksin 3-acetate) and 5,7-dihydroxy-6-methoxy-2,3-dihydroflavonol 3-acetate, a substance not previously characterized. HPLC and (1)H NMR analyses of the propolis and plant extracts indicated smaller amounts of other flavonoids. A survey of propolis samples from 47 apiary sites widely distributed on Kangaroo Island showed that 15 samples from 6 sites were largely sourced from A. paradoxa.

Gingerols of Zingiber officinale enhance glucose uptake by increasing cell surface GLUT4 in cultured L6 myotubes.

In this study we investigate the active constituents of the rhizome of Zingiber officinale, Roscoe (ginger) and determine their activity on glucose uptake in cultured L6 myotubes and the molecular mechanism underlying this action. Freeze-dried ginger powder was extracted with ethyl acetate (1 kg/3 L) to give the total ginger extract, which was then separated into seven fractions, consisting of nonpolar to moderately polar compounds, using a short-column vacuum chromatographic method. The most active fraction (F7) was further purified for identification of its active components. The effect of the extract, fractions, and purified compounds on glucose uptake was evaluated using radioactive labelled 2-[1,2-³H]-deoxy-D-glucose in L6 myotubes. The pungent phenolic gingerol constituents were identified as the major active compounds in the ginger extract enhancing glucose uptake. (S)-[6]-Gingerol was the most abundant component among the gingerols, however, (S)-[8]-gingerol was the most potent on glucose uptake. The activity of (S)-[8]-gingerol was found to be associated primarily with an increase in surface distribution of GLUT4 protein on the L6 myotube plasma membrane, as detected by expression of hemagglutinin epitope-tagged GLUT4 in L6 muscle cells. The enhancement of glucose uptake in L6 rat skeletal muscle cells by the gingerol pungent principles of the ginger extract supports the potential of ginger and its pungent components for the prevention and management of hyperglycemia and type 2 diabetes.

Antiproliferative and antimigratory actions of synthetic long chain n-3 monounsaturated fatty acids in breast cancer cells that overexpress cyclooxygenase-2.

Cyclooxygenase-2 (COX-2) is overexpressed in many human cancers and converts the n-6 polyunsaturated fatty acid (PUFA) arachidonic acid to prostaglandin E(2) (PGE(2)), which drives tumorigenesis; in contrast, n-3 PUFA inhibit tumorigenesis. We tested the hypothesis that these antitumor actions of n-3 PUFA may involve the n-3 olefinic bond. n-3 Monounsaturated fatty acids (MUFAs) of chain length C16-C22 were synthesized and evaluated in MDA-MB-468 breast cancer cells that stably overexpressed COX-2 (MDA-COX-2 cells). Longer chain (C19-C22) n-3 MUFAs inhibited proliferation, activated apoptosis, decreased PGE(2) formation, and decreased cell invasion; C16-C18 analogues were less active. Molecular modeling showed that interactions of Arg120, Tyr355, and several hydrophobic amino acid residues in the COX-2 active site with C19-C22 MUFA analogues were favored. Thus, longer-chain n-3 MUFAs may be prototypes of novel anticancer agents that decrease the formation of PGE(2) in tumor cells that contain high levels of COX-2.

Prenylated cinnamate and stilbenes from Kangaroo Island propolis and their antioxidant activity.

A prenylated cinnamic acid derivative as well as six prenylated tetrahydroxystilbenes were isolated from the ethyl acetate extract of propolis that originated from Kangaroo Island, Australia. Furthermore, six known stilbenes and two known flavanones were also identified from the same sample. Stilbenes are not common in propolis; therefore, Kangaroo Island propolis is considered a unique type of propolis that is rich in prenylated stilbenes. Stilbene propolis from Kangaroo Island showed a stronger scavenging activity towards DPPH free radical than Brazilian green propolis. This strong activity can be explained by the presence of large number of stilbenes, most of them showed strong free radical scavenging activity.

Modulation of P-glycoprotein-mediated anticancer drug accumulation, cytotoxicity, and ATPase activity by flavonoid interactions.

Flavonoids are components of plant foods and of many herbal medicines taken in combination with anticancer drugs. We have examined the potential of flavonoids to affect the accumulation and cytotoxicity of 3 cytotoxic drugs [vinblastine (VLB), daunorubicin (DNR), and colchicine (COL)] that are substrates for the ABC transporter, P-glycoprotein in a vinblastine-resistant T-cell leukemia, CEM/VBL(100), that overexpresses P-glycoprotein. The effects of the flavonoids on accumulation and cytotoxicity of these drugs were different depending on the P-gp substrate used. Most of the 30 flavonoids tested decreased DNR accumulation in the VBL-resistant, but not sensitive, leukemia cells. By contrast, flavonoids that inhibited DNR accumulation enhanced the accumulation of fluorescently labeled vinblastine. None of these flavonoids affected COL accumulation. The effects of the flavonoids on the cytotoxicities of these drugs paralleled their effects on accumulation; the same flavonoids decreased DNR cytotoxicity but increased VLB cytotoxicity and had no effect on COL. Verapamil reversed the accumulation deficit and cytotoxicity of all three P-gp substrates. These effects correlated with the effects of flavonoids on P-gp-ATPase activity. Flavonoids that decreased DNR accumulation stimulated DNR-activated P-gp ATPase, whereas flavonoids that increased fluorescently labeled VLB accumulation inhibited VBL-stimulated P-gp ATPase activity, thereby accounting for the decrease or increase in cancer drug accumulation in resistant cells. We conclude that flavonoids often ingested by cancer patients may have different effects on anticancer drugs and that these findings should be considered in designing future combination treatments for cancer patients.

Potent cytotoxic effects of Calomeria amaranthoides on ovarian cancers.

BACKGROUND: Ovarian cancer remains the leading cause of death from gynaecological malignancy. More than 60% of the patients are presenting the disease in stage III or IV. In spite of combination of chemotherapy and surgery the prognosis stays poor for therapy regimen. METHODS: The leaves of a plant endemic to Australia, Calomeria amaranthoides, were extracted and then fractionated by column chromatography. In vitro cytotoxicity tests were performed with fractions of the plant extract and later with an isolated compound on ovarian cancer cell lines, as well as normal fibroblasts at concentrations of 1-100 µg/mL (crude extract) and 1-10 µg/mL (compound). Cytotoxicity was measured after 24, 48 and 72 hours by using a non-fluorescent substrate, Alamar blue.In vivo cytotoxicity was tested on ascites, developed in the abdomen of nude mice after inoculation with human OVCAR3 cells intraperitoneally. The rate of change in abdomen size for the mice was determined by linear regression and statistically evaluated for significance by the unpaired t test. RESULTS: Two compounds were isolated by chromatographic fractionation and identified by 1H-NMR, 13C-NMR and mass spectrometry analyses, EPD, an α-methylene sesquiterpene lactone of the eremophilanolide subtype, and EPA, an α-methylene carboxylic acid.Cytotoxicity of EPD for normal fibroblasts at all time points IC50 was greater than 10 µg/mL, whereas, for OVCAR3 cells at 48 hours IC50 was 5.3 µg/mL (95% confidence interval 4.3 to 6.5 µg/mL).Both, the crude plant extract as well as EPD killed the cancer cells at a final concentration of 10 µg/mL and 5 µg/mL respectively, while in normal cells only 20% cell killing effect was observed. EPA had no cytotoxic effects.Changes in abdomen size for control versus Cisplatin treated mice were significantly different, P = 0.023, as were control versus EPD treated mice, P = 0.025, whereas, EPD versus Cisplatin treated mice were not significantly different, P = 0.13. CONCLUSIONS: For the first time both crude plant extract from Calomeria amaranthoides and EPD have been shown to have potent anti-cancer effects against ovarian cancer.

Facile and stereoselective synthesis of (Z)-15-octadecenoic acid and (Z)-16-nonadecenoic acid: monounsaturated omega-3 fatty acids.

Facile syntheses of the monounsaturated omega-3 fatty acids, (Z)-15-octadecenoic acid and (Z)-16-nonadecenoic acid, are presented. Commercially available hydroxy fatty acids were esterified and oxidised, followed by the Wittig reaction to introduce the omega-3 olefinic bond; hydrolysis yielded the omega-3 fatty acids in high purity. An examination of different reaction conditions for the Wittig step found that THF as solvent and coupling temperatures of -78 degrees C gave optimal stereoselectivity, affording the omega-3 olefins in Z:E ratios >or= 97:3. The syntheses have overall yields of approximately 43%, and utilise straightforward, robust chemistry, that may be readily scaled up and reproduced. Also presented is a method for accurately determining the double bond geometry and isomeric purity of the fatty acid products using 1H-13C-HSQC NMR and GC-MS, respectively.

Topical 'Sydney' propolis protects against UV-radiation-induced inflammation, lipid peroxidation and immune suppression in mouse skin.

BACKGROUND: Propolis is a honeybee product that has been used in traditional medicine for antioxidant, immune-stimulating, anti-inflammatory and anti-cancer effects. Here, the potential of the topical application of a crude ethanolic extract of Sydney propolis to protect against UV-radiation-induced impairments associated with an increased risk of photocarcinogenesis has been tested in the hairless mouse. METHODS: Solutions providing between 10 and 200 mg/kg propolis were applied to the skin following UV irradiation. The inflammation from exposure to UV (290-400 nm) was quantitated by measurement of increased skinfold thickness; lipid peroxidation was assayed by the induction of thiobarbituric acid reactive species in the skin; immune function was measured by the contact hypersensitivity (CHS) reaction and supported by the changes in epidermal cytokine expression. RESULTS: Propolis protected significantly and dose-dependently against both sunburn oedema and the suppression of CHS, and (at 100 mg/kg) against lipid peroxidation. The overexpression of IL-10 and the depletion of IL-12 characteristic of photoimmune suppression were markedly reduced by propolis. Further, the upregulation of IL-6 was decreased, and the associated induction of haem oxygenase was shown to play a role in propolis skin protection. CONCLUSIONS: Sydney propolis was able to effectively reduce cutaneous inflammation, immunosuppression and lipid peroxidation induced by UV exposure. It is concluded that Sydney propolis might have strong beneficial protective effects against photodamage and skin cancer development in humans.