Martine Cao

Screening of medicinal plants from Reunion Island for antimalarial and cytotoxic activity

AIM OF THE STUDY Nine plants from Reunion Island, selected using ethnopharmacology and chemotaxonomy, were investigated for their potential antimalarial value. MATERIALS AND METHODS Thirty-eight extracts were prepared by maceration using CH(2)Cl(2) and MeOH, and were tested for in vitro activity against the 3D7 and W2 strain of Plasmodium falciparum. The most active extracts were then tested for in vitro cytotoxicity on human WI-38 fibroblasts to determine the selectivity index. Those extracts were also investigated in vivo against Plasmodium berghei infected mice. RESULTS Most active of the extracts tested were the dichloromethane leaves extracts of Nuxia verticillata Lam. (Buddlejaceae), Psiadia arguta Voigt. (Asteraceae), Lantana camara L. (Verbenaceae), the methanol extracts from Aphloia theiformis (Vahl) Benn. (Aphloiaceae) bark, and Terminalia bentzoe L. (Combretaceae) leaves displaying in vitro IC(50) values ranging from 5.7 to 14.1mug/ml. Extracts from Psiadia, Aphloia at 200mg/(kgday) and Teminalia at 50mg/(kgday) also exhibited significant (p<0.0005) parasite inhibition in mice: 75.5%, 65.6% and 83.5%, respectively. CONCLUSION Two plants showed interesting antimalarial activity with good selectivity: Aphloia theiformis and Terminalia bentzoe. Nuxia verticillata still needs to be tested in vivo, with a new batch of plant material.

Metabolomic investigation of the ethnopharmacological use of Artemisia afra with NMR spectroscopy and multivariate data analysis.

ETHNOPHARMACOLOGICAL RELEVANCE Artemisia afra has been used as an infusion to treat malaria throughout the southern parts of Africa, in much the same way as the antimalarial plant Artemisia annua in China. The antiplasmodial activity of purified components from an apolar fraction of Artemisia afra has been shown in the past. No data on the efficacy of the tea infusion prepared from Artemisia afra are however available. OBJECTIVE To investigate the antiplasmodial activity of various extracts of Artemisia afra including an ethnopharmacological prepared sample. To identify polar metabolites in Artemisia afra and Artemisia annua and by using multivariate data analysis investigate the metabolic differences between these species. MATERIALS AND METHODS The antiplasmodial activity of Artemisia afra and Artemisia annua extracts were tested for activity against Plasmodiam falciparum 3D7 (chloroquine-sensitive strain) with chloroquine, quinine and artemisinin as positive controls. Hydrophilic metabolites in Artemisia afra and Artemisia annua were identified directly from the crude extracts through 1D- and 2D-NMR spectra. The NMR spectra were also used to differentiate between the two species using principal component analysis (PCA) for quality control purposes. RESULTS The apolar fractions of both Artemisia afra and Artemisia annua showed activity against P. falciparum while activity was only found in the tea infusion of Artemisia annua. Metabolomic studies using 1D- and 2D-NMR spectroscopy identified 24 semi-polar components in Artemisia afra including three new phenylpropanoids for this species: caffeic acid, chlorogenic acid and 3,5-dicaffeoyl quinic acid. PCA analysis conducted on the samples yielded good separation between the polar extracts of Artemisia afra and Artemisia annua. CONCLUSION These findings shows that there are no in vitro activity in the tea infusion of Artemisia afra and lists the identified metabolites causing the metabolic differences between Artemisia afra and Artemisia annua for quality control purposes.

Antiplasmodial Alkaloids from the Stem Bark of Strychnos malacoclados.

From the stem bark of Strychnos malacoclados, one new bisindole alkaloid, 3-hydroxylongicaudatine Y (1), was isolated along with the known alkaloids vomicine (2), bisnordihydrotoxiferine (3), divarine (4), longicaudatine (5), longicaudatine Y (6), and longicaudatine F (7). All the compounds were tested for their antimalarial activity against the chloroquine-sensitive 3D7 and -resistant W2 strains of Plasmodium falciparum. Longicaudatine was the most active compound with IC₅₀ values of 0.682 and 0.573 µM, respectively. The activity of compounds 1, 3, 4, 6, and 7 against the two strains ranged from 1.191 to 6.220 µM and 0.573 to 21.848 µM, respectively. Vomicine (2), the only monomer isolated, was inactive. The alkaloids of the longicaudatine-type ( 1, 5-7) were more active than those of the caracurine-type (3- 4). The presence of the ether bridge in the molecule seems to increase the antiplasmodial activity. Compounds 1, 5, and 7 were tested against the WI-38 human fibroblast cell line. Longicaudatine was the most cytotoxic compound with an IC₅₀ of 2.721 µM. Longicaudatine F was 40-46 times more active against the two strains of P. falciparum than against the human fibroblasts and was thus considered as the more selective alkaloid. The structures of the compounds were determined based on the analysis of their spectral data.

Application of a new optimization strategy for the separation of tertiary alkaloids extracted from Strychnos usambarensis leaves

The HPLC separation of six alkaloids extracted from Strychnos usambarensis leaves has been developed and optimized by means of a powerful methodology for modelling chromatographic responses, based on three steps, i.e. design of experiments (DoE), independent component analysis (ICA) and design space (DS). This study was the first application of a new optimization strategy to a complex natural matrix. The compounds separated are the isomers isostrychnopentamine and strychnopentamine, 10-hydroxyusambarine and 11-hydroxyusambarine, also strychnophylline and strychnofoline. Three LC parameters have been optimized using a multifactorial design comprising 29 experiments that includes 2 center point replicates. The parameters were the percentage of organic modifiers used at the beginning of a gradient profile which consisted in different proportions of methanol (MeOH) and acetonitrile (MeCN), the gradient time to reach 70% of organic modifiers starting from the initial percentage and the percentage of MeCN found in the mobile phase. Subsequent to the experimental design application, predictive multilinear models were developed and used in order to provide optimal analytical conditions. The optimum assay conditions were: methanol/acetonitrile-sodium pentane sulfonate (pH 2.2; 7.5 mM) (33.4:66.6, v/v) at a mobile phase flow rate of 1 ml/min during a 40.6 min gradient time. The initial organic phase contained 3.7% MeCN and 96.3% MeOH. The method showed good agreement between the experimental data and predictive value throughout the studied parameters space. Improvement of the analysis time and optimized separation for the compounds of interest was possible due to the original and powerful tools applied. Finally, this study permitted the acquisition of isomers profiles allowing the identification of the optimal collecting period of S. usambarensis.

Synthesis and biological evaluation of amino acid methyl ester conjugates of 2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oic acid against the production of nitric oxide (NO)

2-Cyano-3,12-dioxooleana-1,9(11)-dien-28-oic acid (CDDO, 2) was condensed with various amino acid methyl esters at the C-28 carboxylic acid. The new amide conjugates were evaluated for their inhibition of nitric oxide (NO) production in RAW264.7 cells stimulated with interferon-γ (IFNγ). Of these new compounds, CDDO conjugates with alanine, valine, and serine are nearly equipotent to CDDO-ethyl amide (4), a triterpenoid with promising biological activity in numerous disease models. Some of these conjugates also induce the in vitro expression of heme oxygenase-1, and inhibit the proliferation of Panc-1343 pancreatic cells.

17-O-Acetyl,10-hydroxycorynantheol, a Selective Antiplasmodial Alkaloid Isolated from Strychnos usambarensis Leaves

In the course of our investigations on Strychnos usambarensis leaves in order to isolate isostrychnopentamine, the main alkaloid responsible for the antiplasmodial activity of the plant, a new tertiary indolic alkaloid has been isolated: 17-O-acetyl,10-hydroxycorynantheol 1. Its structure was determined by means of spectroscopic and spectrometric methods such as UV, IR, CD, NMR, and ESI-MS. 17-O-acetyl,10-hydroxycorynantheol 1 is one of the most active monomeric indole alkaloids known to date showing an in vitro activity against Plasmodium falciparum close to 5 µM and a high selectivity.

The Rexinoids LG100268 and LG101506 Inhibit Inflammation and Suppress Lung Carcinogenesis in A/J Mice

LG101506 was originally synthesized to overcome some of the undesirable side effects of rexinoids. We compared the anticarcinogenic action of LG101506 and LG100268 and for the first time showed that both drugs are useful for prevention of lung cancer in A/J mice. These molecules markedly reduced tumor number, tumor size, and total tumor burden, when chronically administered to A/J mice that had been initiated with the mutagenic carcinogen, vinyl carbamate. Moreover, LG100268 synergized with the histone deacetylase inhibitor, vorinostat, for prevention of experimental lung cancer and enhanced the effect of carboplatin/paclitaxel for treatment of experimental lung cancer. Both rexinoids diminished the percentage of high-grade, highly malignant adenocarcinomas found at autopsy. In cell culture studies, the rexinoids exhibited potent anti-inflammatory properties at nanoMolar concentrations. These drugs suppressed the ability of lipopolysaccharide to stimulate the synthesis and secretion of nitric oxide and inflammatory cytokines and chemokines, such as IL6, IL1β, CXCL2, and CSF3, in macrophage-like RAW264.7 cells. The present results suggest that LG100268, LG101506, or a related rexinoid may have useful clinical applications in the field of oncology. Cancer Prev Res; 9(1); 105–14. ©2015 AACR.

Abstract 5559: Novel synthetic pyridyl analogues of CDDO-Im with improved stability and their potential use in cancer prevention

Synthetic oleanane triterpenoids are non-cytotoxic, multifunctional drugs with a broad spectrum of applications for prevention and treatment of cancer and for many other chronic diseases. CDDO-Im, 1[2-Cyano-3,12-dioxooleana-1,9(11-dien-28-oyl] imidazole, synthesized more than a decade ago, is one of the most potent triterpenoids known to date with marked anti-inflammatory, cytoprotective, antiproliferative, differentiative and proapoptotic activity on various human and murine tumor cell lines. However, pharmacokinetics of CDDO-Im are not optimal. Therefore, three new pyridyl analogues of CDDO-Im, namely CDDO-3P-Im, CDDO-2P-Im and CDDO-4P-Im, have been synthesized and screened for their possible use as chemopreventive or chemotherapeutic drugs. At low nanomolar concentrations, they were equivalent to CDDO-Im for induction of differentiation in U937 leukemia cells and at higher doses they induced apoptosis. As inflammation and oxidative stress contribute to carcinogenesis, we also assessed their cytoprotective potential. The new compounds suppressed inducible nitric oxide synthase expression in RAW264.7 macrophage-like cells and significantly induced heme oxygenase-1 and NADPH quinone reductase mRNA and protein levels in lung cancer cells as well as in various mouse tissues. Most importantly, pharmacokinetic studies performed in vitro in human plasma and in vivo revealed superior stability for each new analogue. While CDDO-Im was almost completely degraded after 30 min ( 50% still detectable. Six hours after gavage, much higher concentrations of the new derivatives were found in mouse liver, lung, pancreas and kidney in contrast to CDDO-Im. Thus, the new pyridyl analogues have better bioavailability, and because of their potent anti-inflammatory activity and improved stability, they will be tested in vivo in relevant carcinogenesis models. Citation Format: Martine Cao, Evans Onyango, Charlotte Williams, Darlene Royce, Gordon Gribble, Michael Sporn, Karen Liby. Novel synthetic pyridyl analogues of CDDO-Im with improved stability and their potential use in cancer prevention. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5559. doi:10.1158/1538-7445.AM2015-5559