New Metabolites from the Marine Sponge Scopalina hapalia Collected in Mayotte Lagoon.

The biological screening of 44 marine sponge extracts for the research of bioactive molecules, with potential application in the treatment of age-related diseases (cancer and Alzheimer's disease) and skin aging, resulted in the selection of Scopalina hapalia extract for chemical study. As no reports of secondary metabolites of S. hapalia were found in the literature, we undertook this research to further extend current knowledge of Scopalina chemistry. The investigation of this species led to the discovery of four new compounds: two butenolides sinularone J (1) and sinularone K (2), one phospholipid 1-O-octadecyl-2-pentanoyl-sn-glycero-3-phosphocholine (3) and one lysophospholipid 1-O-(3-methoxy-tetradecanoyl)-sn-glycero-3-phosphocholine (4) alongside with known lysophospholipids (5 and 6), alkylglycerols (7-10), epidioxysterols (11 and 12) and diketopiperazines (13 and 14). The structure elucidation of the new metabolites (1-4) was determined by detailed spectroscopic analysis, including 1D and 2D NMR as well as mass spectrometry. Molecular networking was also explored to complement classical investigation and unravel the chemical classes within this species. GNPS analysis provided further information on potential metabolites with additional bioactive natural compounds predicted.

Behavioral avoidance and biological safety of vetiver oil and its constituents against Aedes aegypti (L.), Aedes albopictus (Skuse) and Culex quinquefasciatus Say.

Numerous plant-based repellents are widely used for personal protection against host-seeking mosquitoes. Vitiveria zizanioides (L.) Nash essential oil and its constituents have demonstrated various mosquito repellent activities. In this study, three chemical actions of vetiver oil and five constituents (terpinen-4-ol, α-terpineol, valencene, vetiverol and vetivone) were characterized against Aedes aegypti, Aedes albopictus and Culex quinquefasciatus by using the high-throughput screening assay system (HITSS). Significant contact escape responses in Ae. aegypti and Ae. albopictus to all test compounds at concentrations between 2.5 and 5% were observed. Spatial repellency responses were also observed in some tested mosquito populations depending upon concentrations. The most significant toxic response on mosquitoes was found at the highest concentration, except for vetivone which had no toxic effect on Ae. aegypti and Ae. albopictus. Results on phototoxic and genotoxic hazard revealed that vetiver oil and their constituents showed no phototoxic potential or any significant genotoxic response. In conclusion, vetiver oil and two constituents, valencene and vetiverol, are potentials as active ingredients for mosquito repellency and present no toxicity.

Excito-repellency and biological safety of ß-caryophyllene oxide against Aedes albopictus and Anopheles dirus (Diptera: Culicidae).

The activity of ß-caryophyllene oxide as either a contact or noncontact repellent was evaluated against two laboratory strains (Aedes albopictus and Anopheles dirus) using an excito-repellency test system. N, N-Diethyl-3-methylbenzamide (DEET) was used as a standard reference baseline for comparative purposes. ß-Caryophyllene oxide and DEET were tested at concentrations of 0.1, 0.25, 0.5 and 1.0% (v/v). In addition, the phototoxic and genotoxic effects of ß-caryophyllene oxide were investigated on Balb/c 3T3 mouse fibroblasts (3T3-L1) and Chinese hamster ovary cell line (CHO-K1). The results demonstrated that the higher concentrations of test compounds (0.5 and 1.0%) produced greater behavioral responses. Aedes albopictus was more sensitive to ß-caryophyllene oxide than An. dirus. Moderate avoidance response rates (25-56% escape) of Ae. albopictus at 0.5% and 1.0% ß-caryophyllene oxide were observed in contact and noncontact trials compared with low response rates from An. dirus (26-31% escape). DEET at ≤1% displayed lower irritancy and repellency (1-38%) than ß-caryophyllene oxide when tested against the two mosquito species. Knockdown responses (37%) were only observed in An. dirus exposed to 1% ß-caryophyllene oxide in the contact trial. ß-Caryophyllene oxide did not show any phototoxic potential (PIF= 0.38) nor was there any significant genotoxic response as indicated by no increase in micro-nucleated cells with or without metabolic activation. ß-Caryophyllene oxide could be considered as a safe repellent, effective against mosquitoes.

Trypanocidal and leishmanicidal activity of six limonoids.

Six limonoids [kotschyienone A and B (1, 2), 7-deacetylgedunin (3), 7-deacetyl-7-oxogedunin (4), andirobin (5) and methyl angolensate (6)] were investigated for their trypanocidal and leishmanicidal activities using bloodstream forms of Trypanosoma brucei and promastigotes of Leishmania major. Whereas all compounds showed anti-trypanosomal activity, only compounds 1-4 displayed anti-leishmanial activity. The 50% growth inhibition (GI50) values for the trypanocidal and leishmanicidal activity of the compounds ranged between 2.5 and 14.9 µM. Kotschyienone A (1) was found to be the most active compound with a minimal inhibition concentration (MIC) value of 10 µM and GI50 values between 2.5 and 2.9 µM. Only compounds 1 and 3 showed moderate cytotoxicity against HL-60 cells with MIC and GI50 values of 100 µM and 31.5-46.2 µM, respectively. Compound 1 was also found to show activity against intracellular amastigotes of L. major with a GI50 value of 1.5 µM. The results suggest that limonoids have potential as drug candidates for the development of new treatments against trypanosomiasis and leishmaniasis.

Excito-repellent activity of ß-caryophyllene oxide against Aedes aegypti and Anopheles minimus.

Contact irritant and non-contact repellent activities of ß-caryophyllene oxide were evaluated against laboratory strains of female Aedes aegypti (USDA strain), a major arbovirus vector and Anopheles minimus (KU strain), a major malaria parasite vector, compared with the synthetic repellent DEET, using an excito-repellency test system. ß-caryophyllene oxide and DEET were tested at concentrations of 0.1, 0.25, 0.5 and 1.0% (v/v). Anopheles minimus was found to be more sensitive to ß-caryophyllene oxide than that of Ae. aegypti and exhibited high avoidance response rates (86-96% escape) at 0.5% and 1.0% concentrations in contact and non-contact trials compared with Ae. aegypti (22-59% escape). However, at the same concentrations, DEET displayed lower irritancy and repellency capacities against these two mosquito species (range 0-54% escape) compared to ß-caryophyllene oxide. The analysis of escape responses showed significant differences between mosquito species at all concentrations (P < 0.05) except for 0.1%. For both species, there were significant differences in irritant and repellent responses between ß-caryophyllene oxide and DEET at higher concentrations (0.5 and 1.0%).

Design and synthesis of simplified speciophylline analogues and ß-carbolines as active molecules against Plasmodium falciparum.

A structure-activity relationship study of active molecules against chloroquine-resistant Plasmodium falciparum K1 strain is reported. Structurally simplified analogues of antiplasmodial active alkaloids presented similar levels of activity as their corresponding natural products extracted from Guiera senegalensis and Mitragyna inermis with IC50 values on chloroquine-resistant P. falciparum K1 strain of up to 10.6 µM for spirooxindoles and 13.8 µM for ß-carbolines. The identification of such simpler and cheaper structural analogues is crucial to efficiently study these natural products' action mode as well as developing new cures against malaria.

Bioassay-guided isolation and UHPLC-DAD-ESI-MS/MS quantification of potential anti-inflammatory phenolic compounds from flowers of Inula montana L.

ETHNOPHARMACOLOGICAL RELEVANCE: Flowers of Inula montana L. (Asteraceae), commonly known as "Arnica de Provence", are used in the traditional medicine of Provence in France with the same indication as Arnica montana, for the relief of bruises, as an anti-inflammatory agent. AIMS OF THE STUDY: The aim of our study is to evaluate its anti-inflammatory properties and to justify its traditional uses. Its potential valorization is evaluated in order to propose Inula montana as an alternative to Arnica montana. MATERIALS AND METHODS: Bio-guided fractionation of ethanolic extract allowed the isolation of compounds responsible of the inhibition of NO production. The fractionation was realized using chromatographic techniques and structure elucidation was conducted by ESI-MS and NMR spectral data. Anti-inflammatory effect of ethanolic extract, different fractions and isolated pure compounds was studied in vitro on immortalized mouse macrophages RAW 264.7. An analytical UHPLC-DAD-ESI-MS/MS method was developed for the identification of these compounds in the herbal drug. This UHPLC-DAD method was validated and was used to compare the phenolic profile and content in plant material from the two collection sites: Bonnieux and Merindol. RESULTS: Eleven compounds were identified by UHPLC-MS. Chlorogenic acid (1), Luteolin (2), Nepetin (3), 3,5-O-Dicaffeoylquinic acid (4), 1,5-O-Dicaffeoylquinic acid (5), Nepitrin (6), Hispiduloside (7) and Jaceosid (8) were isolated and identified by NMR. Compounds 9, 10 and 11 were confirmed to be 6-Hydroxykaempferol 3,7-dimethyl ether, Hispidulin and Chrysosplenol C, respectively by comparing retention times and MS/MS data with those of the authentic substances. Six compounds: 1 and 4-8 are reported for the first time in Inula montana L. Compounds 2-8 showed promising anti-inflammatory activity with the release of NO with IC50 value < 7 µM. The UHPLC-DAD method of quantification of three major bioactive compounds (1, 3 and 5) was validated. CONCLUSION: Flowers extracts and isolated compounds present promising anti-inflammatory activity which provides a scientific basis for the traditional use of Inula montana and may be proposed in the same indications as Arnica montana. The developed and validated simple, accurate and rapid UHPLC method can be used for the quality control of the herbal drug.

Bioactive Seco-Lanostane-Type Triterpenoids from the Roots of Leplaea mayombensis.

Fractionation of the ethyl acetate-soluble extract of the roots of Leplaea mayombensis afforded two new 3,4-seco-lanostane-type triterpenoids, leplaeric acids A and B (1, 2), the new lanostane-type triterpenoid leplaeric acid C (3), and six known natural products (5-10). Derivatization of the main constituent, 1, afforded the dimethyl ester 4, the monoamide 11, and diamide 12 for SAR studies. The structures of these compounds were established through spectroscopic methods, and a single-crystal X-ray diffraction analysis was used to confirm the relative configuration of compound 1. These lanostane derivatives are unique since they are the first C-21-oxygenated lanostanes isolated from plant sources. Preliminary biological assays against the MDA MB 231 breast cancer cell line showed that compounds 1, 2, 4, and 11 have modest cytotoxic activity. Compound 2 was the most active, with an IC50 of 55 ± 7 µM. From these results, the amides (11, 12) derived from triterpenoid 1 were found to be less active than the derived esters (2, 4).

New sesquiterpene acid and inositol derivatives from Inula montana L.

A phytochemical investigation of the ethanol extract of leaves and flowers of Inula montana L. led to the isolation of one new sesquiterpene acid called Eldarin (1) and four new inositol derivatives, Myoinositol,1,5-diangelate-4,6-diacetate (2), Myoinositol,1,6-diangelate-4,5-diacetate (3), Myoinositol-1-angelate-4,5-diacetate-6-(2-methylbutirate) (4), Myoinositol-1-angelate-4,5-diacetate-6-isovalerate (5) isolated for the first time, along with eleven known compounds described for the first time in Inula montana, 1ß-Hydroxyarbusculin A (6), Artemorin (7), Santamarin (8), Chrysosplenol C (9), 6-Hydroxykaempferol 3,7-dimethyl ether (10), Reynosin (11), Calenduladiol-3-palmitate (12), Costunolide (13), 4-Hydroxy-3,5-dimethoxybenzenemethanol (14), 9ß-Hydroxycostunolide (15) and Hispidulin (16). Structural elucidation has been carried out by spectral methods, such as 1D and 2D NMR, IR, UV and HR-ESI-MS. These compounds have been tested in vitro for anti-inflammatory and cytotoxic activity on macrophages RAW 264.7. As a result, compounds 2, 3, 7, 13, 14, 15 and 16 showed a release of NO with IC50 value <30µM on macrophages.

Chemical profiling of the tuber of Stephania cambodica Gagnep. (Menispermaceae) and analytical control by UHPLC-DAD.

A new aporphine glycoside (1), named 'angkorwatine', and eight known alkaloids: oblongine (2), stepharine (3), asimilobine-ß-d-glucopyranoside (4), isocorydine (5), tetrahydropalmatine (THP) (6), jatrorrhizine (7), palmatine (PAL) (8), and roemerine (ROE) (9) were simultaneously isolated from the tuber of Stephania cambodica. The development and validation of UHPLC-DAD method was carried out for the quantification of marker compounds (PAL, ROE, THP) of S. cambodica. In addition to good selectivity and linearity (r2 > 0.997), trueness, precision, and accuracy of the method did not exceed the acceptance limit of ±10% for ROE, THP and ±20% for PAL. Consequently, this method is able to provide accurate results between 1.39-4.18 µg/mL, 2.01-30.72 µg/mL, and 4.29-64.42 µg/mL for PAL, ROE, and THP, respectively. This study shows that the validated UHPLC method is a rapid, innovative and effective analytical approach to control quality of tubers of S. cambodica and to regulate the usage of this plant in traditional medicine.

Comparative Excito-Repellency of Three Cambodian Plant-Derived Extracts Against Two Mosquito Vector Species, Aedes aegypti and Anopheles minimus.

A study of the behavioral responses of Aedes aegypti and Anopheles minimus to 3 Cambodian plant extracts at 3 different concentrations (1%, 2.5%, and 5%) was performed using an excito-repellency test system. These 3 plants were Strophanthus scandens, Capparis micracantha, and Dioscorea hispida, selected according to traditional healer's knowledge, bibliographic studies and market surveys. Results showed that S. scandens leaves' hexane extract was the only one to exert repellency against Ae. aegypti with 23.3% of escaped mosquitoes at a concentration of 5%. Capparis micracantha was responsible for an irritant activity against An. minimus with 20.2% of escaped mosquitoes at a concentration of 2.5% and 22.8% escaping at a concentration of 5%. Dioscorea hispida showed an irritant activity on both mosquito species with 23.2% of escaped Ae. aegypti at a concentration of 5% and about 20% of escaped An. minimus at 2.5% and 5%. This is the first report on the irritant and repellent activities of S. scandens , D. hispida , and C. micracantha against mosquito species.

In vitro antiplasmodial activity of cepharanthine.

BACKGROUND: New classes of anti-malarial drugs are needed to control the alarming Plasmodium falciparum resistance toward current anti-malarial therapy. The ethnopharmacological approach allows the discovery of original chemical structures from the vegetable biodiversity. Previous studies led to the selection of a bisbenzylisoquinoline, called cepharanthine and isolated from a Cambodian plant: Stephania rotunda. Cepharanthine could exert a mechanism of action different from commonly used drugs. Potential plasmodial targets are reported here. METHODS: To study the mechanism of action of cepharanthine, a combined approach using phenotypic and transcriptomic techniques was undertaken. RESULTS: Cepharanthine blocked P. falciparum development in ring stage. On a culture of synchronized ring stage, the comparisons of expression profiles showed that the samples treated with 5 µM of cepharanthine (IC90) were significantly closer to the initial controls than to the final ones. After a two-way ANOVA (p-value < 0.05) on the microarray results, 1,141 probes among 9,722 presented a significant differential expression.A gene ontology analysis showed that the Maurer's clefts seem particularly down-regulated by cepharanthine. The analysis of metabolic pathways showed an impact on cell-cell interactions (cytoadherence and rosetting), glycolysis and isoprenoid pathways. Organellar functions, more particularly constituted by apicoplast and mitochondrion, are targeted too. CONCLUSION: The blockage at the ring stage by cepharanthine is described for the first time. Transcriptomic approach confirmed that cepharanthine might have a potential innovative antiplasmodial mechanism of action. Thus, cepharanthine might play an ongoing role in the progress on anti-malarial drug discovery efforts.

Quantitative analysis of cepharanthine in plasma based on semiautomatic microextraction by packed sorbent combined with liquid chromatography.

The spread of Plasmodium falciparum resistance toward most of the used drugs requires new antimalarial compounds. Taking advantage of the biodiversity, the ethnopharmacological approach opens the way for the discovery and the characterization of potent original molecules. Previous works led to the selection of a bisbenzylisoquinoline, cepharanthine, extracted from Stephania rotunda, which is mainly present in Cambodia. A sensitive and selective liquid chromatography method has been developed for the determination of cepharanthine in mouse plasma. The method involved a semiautomated microextraction by packed sorbent (MEPS) using 4 mg of solid phase silica-C8 sorbent. LC separation was performed on a Kinetex XB-C18 column (2.6 µm) with a mobile phase of acetonitrile containing formic acid and 10 mM ammonium formate buffer pH 3.5. Data were acquired at 282 nm with a diode array detector. The drug/internal standard peak area ratios were linked via linear relationships to plasma concentrations (75-2,000 ng/mL). Precision was below 5% and accuracy was 99.0-102%. Extraction recovery of cepharanthine was 56-58%. The method was successfully used to determine the pharmacokinetic profile of cepharanthine in healthy and Plasmodium berghei infected mice. The infection did not impact pharmacokinetic parameters of cepharanthine.

Ethnobotany, phytochemistry and pharmacology of Stephania rotunda Lour.

ETHNOPHARMACOLOGICAL RELEVANCE: Stephania rotunda Lour. (Menispermaceae) is an important traditional medicinal plant that is grown in Southeast Asia. The stems, leaves, and tubers have been used in the Cambodian, Lao, Indian and Vietnamese folk medicine systems for years to treat a wide range of ailments, including asthma, headache, fever, and diarrhoea. AIM OF THE REVIEW: To provide an up-to-date, comprehensive overview and analysis of the ethnobotany, phytochemistry, and pharmacology of Stephania rotunda for its potential benefits in human health, as well as to assess the scientific evidence of traditional use and provide a basis for future research directions. MATERIAL AND METHODS: Peer-reviewed articles on Stephania rotunda were acquired via an electronic search of the major scientific databases (Pubmed, Google Scholar, and ScienceDirect). Data were collected from scientific journals, theses, and books. RESULTS: The traditional uses of Stephania rotunda were recorded in countries throughout Southeast Asia (Cambodia, Vietnam, Laos, and India). Different parts of Stephania rotunda were used in traditional medicine to treat about twenty health disorders. Phytochemical analyses identified forty alkaloids. The roots primarily contain l-tetrahydropalmatine (l-THP), whereas the tubers contain cepharanthine and xylopinine. Furthermore, the chemical composition differs from one region to another and according to the harvest period. The alkaloids exhibited approximately ten different pharmacological activities. The main pharmacological activities of Stephania rotunda alkaloids are antiplasmodial, anticancer, and immunomodulatory effects. Sinomenine, cepharanthine, and l-stepholidine are the most promising components and have been tested in humans. The pharmacokinetic parameters have been studied for seven compounds, including the three most promising compounds. The toxicity has been evaluated for liriodenine, roemerine, cycleanine, l-tetrahydropalmatine, and oxostephanine. CONCLUSION: Stephania rotunda is traditionally used for the treatment of a wide range of ailments. Pharmacological investigations have validated different uses of Stephania rotunda in folk medicine. The present review highlights the three most promising compounds of Stephania rotunda, which could constitute potential leads in various medicinal fields, including malaria and cancer.

In vitro and in vivo combination of cepharanthine with anti-malarial drugs.

BACKGROUND: Stephania rotunda is used by traditional health practitioners in Southeast Asia to treat a wide range of diseases and particularly symptoms related to malaria. Cepharanthine (CEP) is an alkaloid isolated from this plant with potential innovative antiplasmodial activity. The analysis of interactions between antiplasmodial drugs is necessary to develop new drugs combinations to prevent de novo emergence of resistance. The objective of this study was to evaluate the anti-malarial activity of CEP in combination with usual anti-malarial compounds, both in vitro and in vivo. METHODS: A fixed ratio method using the isotopic micro test was performed on the chloroquine-resistant plasmodial strain W2 to build isobolograms from eight CEP-based combinations with standard anti-malarial drugs. The efficacy of two combinations was then evaluated in the BALB/c mouse infected with Plasmodium berghei ANKA strain. RESULTS: In vitro, efficiency gains were observed when CEP was combined with chloroquine (CQ), lumefantrine (LUM), atovaquone (ATO), piperaquine (PPQ) and particularly monodesethylamodiaquine (MdAQ), whereas an antagonistic interaction was observed with dihydroartemisinin (DHA) and mefloquine (MQ). In vivo, the combination of CEP with CQ or amodiaquine (AQ) improved significantly the survival of mice and extended the delay for parasitic recrudescence. CONCLUSION: All these observations suggest that CEP could be an interesting lead compound in the development of a combination therapy against malaria.

In vitro cytotoxic and anticlastogenic activities of saxifragifolin B and cyclamin isolated from Cyclamen persicum and Cyclamen libanoticum.

CONTEXT: The genus Cyclamen L. (Primulaceae) is rich in saponins known to have interesting biological activities. OBJECTIVE: To isolate saxifragifolin B and cyclamin, two triterpene saponins, from Cyclamen libanoticum Hildebr and Cyclamen persicum Mill, and to assess their cytotoxic, clastogenic/aneugenic, and anticlastogenic effects, as well as antioxidant potential. MATERIALS AND METHODS: Saxifragifolin B and cyclamin were tested for their cytotoxicity against SK-BR-3, HT-29, HepG2/3A, NCI-H1299, BXPC-3, 22RV1, and normal DMEM cell lines using WST-1 assay. Their clastogenic/aneugenic activities and anticlastogenic effects against the anticancer drug mitomycin C were assessed by the in vitro micronucleus assay in CHO cells. Their antioxidant capacities were determined using Fe(2+)-chelating and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging assays. RESULTS: Both saponins were described for the first time in Cyclamen libanoticum. They showed strong cytotoxic activities against the tested cancer cell lines. Saxifragifolin B was found to be 56- and 37-times more active than mitomycin C against breast adenocarcinoma (SK-BR-3) and lung carcinoma (NCI-H1299), respectively. Also, saxifragifolin B did not induce micronuclei formation and prevented cells from mitomycin C clastogenic effect. Cyclamin induced a significant increase of micronucleated cells after metabolic activation with S9 mix, and did not possess any anticlastogenic activity. Both molecules exhibited low antioxidant activities as compared to reference compounds. DISCUSSION AND CONCLUSIONS: This study showed the remarkable cytotoxic activity of saxifragifolin B, especially against breast adenocarcinoma and lung carcinoma and its chemoprotective activity against mitomycin C. Thus, saxifragifolin B could be suggested as a potential cytotoxic drug with a preventive effect against possible exposures to genotoxic agents.

In vitro antiplasmodial activity of some medicinal plants of Burkina Faso.

Malaria remains a major public health problem due to the emergence and spread of Plasmodium falciparum drug resistance. There is an urgent need to investigate new sources of antimalarial drugs which are more effective against Plasmodium falciparum. One of the potential sources of antimalarial drugs is traditional medicinal plants. In this work, we studied the in vitro antiplasmodial activity of chloromethylenic, methanolic, and MeOH/H2O (1/1) crude extracts and decoction obtained from eight medicinal plants collected in Burkina Faso and of total alkaloids for five plants. Extracts were evaluated in vitro for efficacy against Plasmodium falciparum strain K1, which is resistant to chloroquine, pyrimethamine and proguanil using the fluorescence-based SYBR Green I assay. The antiproliferative activity on human-derived hepatoma cell line HepG2 and Chinese hamster ovary (CHO) cells was evaluated using the 3-[4,5-dimethylthyazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) test in order to determine the selectivity index. Among the plant extracts tested for in vitro antiplasmodial activity, 16 were considered to be inactive (with IC50 > 10 µg/ml), six showed a moderate activity (5 < IC50 ≤ 10 µg/ml), and six were found to have a good in vitro activity with IC50 value ≤ 5 µg/ml. The highest antiplasmodial activity was found for extracts from: the alkaloid leaf extract and the chloromethylenic extracts of Combretum fragrans (IC50 = 3 µg/ml, IC50 = 5 µg/ml), the total alkaloids and the chloromethylenic leaf extracts of Combretum collinum (IC50 = 4 µg/ml), the MeOH/H2O leaf extract of Terminalia avicennioides (IC50 = 3.5 µg/ml), and the alkaloid leaf extract of Pavetta crassipes (IC50 = 5 µg/ml). Three other extracts showed moderate antiplasmodial activity (5 < IC50 ≤ 10 µg/ml): Terminalia avicennioides and Combretum fragrans methanolic extracts and Acacia kirkii alkaloid leaf extract (IC50 = 6.5, 9 and 10 µg/ml respectively). The Terminalia avicennioides crude MeOH/H2O (80:20 v/v) extract of the leaves was submitted to a successive liquid/liquid extraction with ethylacetate and n-butanol respectively. The extracts were investigated for in vitro antiplasmodial activity and antioxidant properties using DPPH(·), ABTS(+) and FRAP methods. The ethylacetate extract showed the best antiplasmodial activity (7 µg/ml) and the active constituent was isolated as ellagic acid by bioguided fractionation with an IC50 = 0.2 µM on Plasmodium falciparum and SI = 152. Besides, Terminalia avicennioides leaf extract and ellagic acid showed a good antioxidant activity. Our finding confirms the importance of investigating the antimalarial activity of plant species used in traditional medicine. Overall, two plants belonging to the Combretaceae family, Combretum fragrans and Combretum collinum appeared to be the best candidates and will be further investigated for their antiplasmodial properties, in order to isolate the molecules responsible for the antiplasmodial activity.

Cytotoxic withanolides from the leaves of Moroccan Withania frutescens.

CONTEXT: Withania species are a rich source of interesting phytochemical substances (withanolides) which have shown several biological properties. OBJECTIVE: To investigate the cytotoxic potential of Withania frutescens (L.) Pauquy (Solanaceae) leaf extracts and isolated active compounds against cultured tumor cell lines. MATERIALS AND METHODS: The crude methanol extract of W. frutescens leaves was partitioned with dichloromethane, ethyl acetate and n-butanol. MeOH extract and its fractions were tested for their cytotoxic activity against cancer cell lines (HepG2 and HT29) using the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay. Bioassay-guided fractionation was performed for the active CH2Cl2 fraction employing column chromatography and preparative high-performance liquid chromatography. Structural elucidation of the isolated active compounds was carried out mainly by 1D and 2D NMR and mass spectrometry. The compounds were then tested for their cytotoxic activity. RESULTS: The CH2Cl2 fraction was the most active against HT29 cell line. The fractionation procedure resulted in the isolation of 4ß,17α,27-trihydroxy-1-oxo-22-R-witha-2,5,24-trienolide (1), 5ß,6ß-epoxy-4ß,17α,27-trihydroxy-1-oxowitha-2,24-dienolide (2) and 2,3-dihydroxywithaferin A-3ß-O-sulfate (3). The latter exhibited the strongest cytotoxic activity against HT29 cancer cell lines (IC50 of 1.78 ± 0.09 µM) which was comparable to that of 5-fluorouracil (5-FU) used as the positive antimitotic control. DISCUSSION AND CONCLUSION: Compounds 2 and 3 were isolated from W. frutescens for the first time. Data obtained suggest that the sulfated steroidal lactone (3) can be considered as a compound with potential application in the new anticancer drugs development field.

HPLC analysis of Stephania rotunda extracts and correlation with antiplasmodial activity.

Stephania rotunda (Menispermaceae), a creeper commonly found in the mountainous areas of Cambodia, has been mainly used for the treatment of fever and malaria. Thus, the aim of this study is to investigate the chemical composition and antiplasmodial activity of different samples of S. rotunda and compare their antiplasmodial activity with their alkaloid content. Sixteen samples from different parts (roots, stem, and tuber) of S. rotunda were collected from four regions of Cambodia (Battambang, Pailin, Siem Reap, and Kampot). Reversed-phase HPLC was used to determine the content of three bioactive alkaloids (cepharanthine, tetrahydropalmatine, and xylopinine). These three alkaloids have been found in all samples from Battambang and Pailin (samples I-IX), whereas only tetrahydropalmatine was present in samples from Siem Reap and Kampot (samples X-XVI). The analyzed extracts were evaluated for their antiplasmodial activity on W2 strain of Plasmodium falciparum. Among them, 13 extracts were significantly active with inhibitory concentration 50 (IC(50) ) from 1.2 to 3.7 µg/mL and 2 extracts were moderately active (IC(50) = 6.1 and 10 µg/mL, respectively), whereas sample XI was not active (IC(50) = 19.6 µg/mL). A comparison between antiplasmodial activity and concentration of the three bioactive alkaloids in S. rotunda extracts has been realized.

New antiplasmodial alkaloids from Stephania rotunda.

ETHNOPHARMACOLOGICAL RELEVANCE: Stephania rotunda Lour. (Menispermaceae) is a creeper growing in many countries of Asia and commonly found in the mountainous areas of Cambodia. As a folk medicine, it has been mainly used for the treatment of fever and malaria. The pharmacological activity is mostly due to alkaloids. Thus the aim of this study is to isolate new bioactive alkaloids from Stephania rotunda and to evaluate their in vitro antiplasmodial activity. MATERIALS AND METHODS: Alkaloids were isolated and identified from dichloromethane and aqueous extracts using a combination of flash chromatography, high performance liquid chromatography, mass spectrometry and nuclear magnetic resonance. The purified compounds were tested for in vitro antiplasmodial activity on chloroquine-resistant W2 strain of Plasmodium falciparum. RESULTS: A new aporphine alkaloid named vireakine (2) along with two known alkaloids stephanine (1) and pseudopalmatine (8), described for the first time in Stephania rotunda, and together five known alkaloids tetrahydropalmatine (3), xylopinine (4), roemerine (5), cepharanthine (6) and palmatine (7) were isolated and identified. The structure of the new alkaloid was established on the basis of 1D and 2D NMR experiments and mass spectrometry. The compounds were evaluated for their in vitro antiplasmodial and cytotoxic activities. All tested compounds showed significant antiplasmodial activities with IC(50) ranged from 1.2 µM to 52.3 µM with a good selectivity index for pseudopalmatine with IC(50) of 2.8 µM against W2 strain of Plasmodium falciparum and IC(50)>25 µM on K562S cells. CONCLUSIONS: This study provides evidence to support the use of Stephania rotunda for the treatment of malaria and/or fever by the healers. Alkaloids of the tuber exhibited antiplasmodial activity and particularly cepharanthine and pseudopalmatine.