Antiplasmodial activity of Heinsia crinita (Rubiaceae) and identification of new iridoids.

ETHNOPHARMACOLOGICAL RELEVANCE: Heinsia crinita is used in traditional medicine for the treatment of febrile illness and erectile dysfunction. Its stem bark powder is found in some peripheral markets in the Democratic Republic of the Congo (DRC) as a remedy against malaria. Investigations were conducted on crude extracts of leaves, fruits and stem barks in view to validate their use and to determine which plant part possesses the best antiplasmodial properties. MATERIALS AND METHODS: Different plant parts were extracted with methanol, ethanol and dichloromethane. Based on the preliminary assays, the dichloromethane extract of the stem bark was subjected to fractionation using preparative HPLC system and column chromatography. This step led to the isolation of two new iridoids which had their structures elucidated by NMR, UV, MS and FT-IR spectroscopic techniques. Extracts and pure compounds were tested in vitro against the 3D7 strain of Plasmodium falciparum. The inhibition of the parasite growth was evaluated in vitro by colorimetric method (p-LDH assay) and their cytotoxicity evaluated in vitro against the human non-cancer fibroblast cell line (WI38) through WST1 assay. The in vivo antiplasmodial activity was assessed by the inhibition of Plasmodium berghei growth in infected mice treated with the ethanol extract of H. crinita stem bark at the concentrations of 200 and 300mg/Kg/day per os, using a protocol based on the 4-d suppressive test of Peters and compared to a non-treated negative control group of mice (growth =100%). Finally the antioxidant activity of the same extract was evaluated using ABTS, DPPH and cell-based assays. RESULTS: A moderate in vitro antiplasmodial activity was observed for the dichloromethane extract of the stem bark of H. crinita (IC50 =29.2±1.39µg/mL) and for the two new iridoids, lamalbide 6, 7, 8- triacetate (IC50 =16.39±0.43µg/mL) as well as for its aglycone lamiridosin 6, 7, 8-triacetate (IC50 =0.44.56±1.12µg/mL). The ethanolic stem bark extract (200 and 300mg/kg/day, oral route) showed a moderate in vivo antimalarial activity in Plasmodium berghei-infected mice with 27.84±2.75% and 48.54±3.76% of inhibition of the parasite growth, respectively (p<0.01).). This extract displayed high cellular antioxidant activity using dichlorofluorescein-diacetate (DCFDA) on HL-60 monocytes. These crude extracts and pure compounds tested at the higher concentration of 100µg/mL did not show any cytotoxicity against WI38 cells. CONCLUSIONS: The results showed that H. crinita extracts possess antimalarial activity and contain some unusual iridoids with moderate antiplasmodial activity, therefore justifying to some extent its traditional use by the local population in DRC for this purpose. This is the first report of the isolation and antiplasmodial activity of these two new iridoids.

Antiplasmodial, anti-inflammatory and cytotoxic activities of various plant extracts from the Mascarene Archipelago.

AIM OF THE STUDY: Antiplasmodial activity, inhibition of nitric oxide (NO) overproduction, and anti-proliferative activity were investigated in vitro to evaluate the bioactive potential of the traditional pharmacopoeia of the Mascarene Archipelago, which is known for its biodiversity and for the richness of its endemic flora. MATERIALS AND METHODS: A total of 45 methanol (MeOH) and dichloromethane (DCM) extracts were prepared from 19 plant species collected on Réunion and Mauritius Islands. Ninety-six-well microplate assays were performed on chloroquine sensitive Plasmodium falciparum 3D7 strain, on LPS-stimulated Raw 264.7 murine macrophages and on A-549, DLD-1 and WS1 human cells. Activity was evaluated through spectrophotometric methods. RESULTS: Activity was attributed to plant extracts expressing IC(50)<50µg/ml for antiplasmodial response, IC(50)<100µg/ml for cytotoxicity, and IC(50)<130µg/ml for anti-inflammatory reaction. The majority of the extracts tested (69%) exhibited potency in at least one of these three types of activity. This is the first report describing promising antiplasmodial activity (IC(50)<15µg/ml) for Psiadia dentata DCM extract and Terminalia bentzoe MeOH bark extract. NO inhibition assay revealed seven interesting plants, described for the first time as anti-inflammatory: Aphloia theiformis, Buddleja salviifolia, Eupatorium riparium, Hiptage benghalensis, Psiadia arguta, Psiadia dentata, and Scutia commersonii. Finally, anti-proliferative activity was observed for two endemic species, Geniostoma borbonicum and Nuxia verticillata. CONCLUSION: Using the criterion of endemism as part of the criteria for traditional medicinal use raises the chances of finding original active principles. In our case, 86% of the endemic plants tested displayed pharmacological interest.

Antiplasmodial and cytotoxic activities of Rwandan medicinal plants used in the treatment of malaria.

AIM OF THE STUDY: In our study, methanol, dichloromethane and aqueous extracts of 13 Rwandan medicinal plants used in the treatment of malaria were tested for in vitro antiplasmodial activity. MATERIALS AND METHODS: The growth inhibition of chloroquine-sensitive Plasmodium falciparum strain (3D7) was evaluated using the measurement of lactate dehydrogenase activity. The active extracts were also tested against the chloroquine-resistant Plasmodium falciparum strain (W2) and for cytotoxicity assay using human normal foetal lung fibroblasts (WI-38). RESULTS: The majority of the plants tested showed an antiplasmodial activity and the best results were observed with dichloromethane leaf and flower extracts of Tithonia diversifolia, leaf extract of Microglossa pyrifolia and root extract of Rumex abyssinicus, methanol leaf extract of Fuerstia africana, root bark extracts of Zanthoxylum chalybeum and methanol bark extract of Terminalia mollis. Those extracts were active (IC(50)<15mug/ml) on both chloroquine-sensitive and resistant strains of Plasmodium falciparum. Zanthoxylum chalybeum, Solanecio mannii and Terminalia mollis presented the best selectivity index. CONCLUSIONS: The traditional use of most of the plant evaluated was confirmed by the antiplasmodial test. This study revealed for the first time the antiplasmodial activity of two plants: Terminalia mollis and Rumex abyssinicus.

Screening of 14 alkaloids isolated from Haplophyllum A. Juss. for their cytotoxic properties.

Further to a systematic chemotaxonomic study of Uzbek Haplophyllum A. Juss. plants selected on ethnopharmacological data, 14 alkaloids were screened for their cytotoxic properties. As a first selection for interesting compounds, each alkaloid was tested against two human cancer cell lines (HeLa and HCT-116), using WST-1 reagent. Of the 14 alkaloids, 5 were cytotoxic when tested against the HeLa line with an IC50 < 100 microM. These five compounds consisted of three furoquinolines: skimmianine; haplopine and gamma-fagarine and two pyranoquinolones: flindersine and haplamine. Only haplamine was active against the HCT-116 line. The cytotoxic properties of these five alkaloids were further investigated against five additional human cancer cell lines. Their structure-activity relationships will be discussed. Of these five pre-selected alkaloids, only haplamine showed significant cytotoxic activity against all the tested cell lines. This is the first report of the cytotoxic activity of haplamine. Finally, this pyranoquinolone alkaloid was tested here against 14 different cancer cell lines and against normal skin fibroblasts.

Reversal of chloroquine and mefloquine resistance in Plasmodium falciparum by the two monoindole alkaloids, icajine and isoretuline.

Eight naturally occurring monoindole alkaloids were evaluated in vitro for their ability to inhibit Plasmodium falciparum growth and, in drug combination, to reverse the resistance of a chloroquine-resistant strain of Plasmodium falciparum. None of these indole alkaloids has significant intrinsic antiplasmodial activity (IC(50) > 10 microM or 5 microg/ml). Nevertheless, three alkaloids (icajine, isoretuline and strychnobrasiline) did reverse chloroquine resistance at concentrations between 2.5 and 25 microg/ml (IF of 12.82 for isoretuline on W2 strain). The Interaction Factor (IF) equals 2, < 2, or > 2 for additive, antagonistic or synergistic effects of alkaloids on chloroquine inhibition, respectively. Icajine and isoretuline were also assessed in vitro for their mefloquine potentiating activity on a mefloquine-resistant strain of Plasmodium falciparum. Only icajine proved to be synergistic with mefloquine (IF = 15.38).

Strychnogucines A and B, two new antiplasmodial bisindole alkaloids from Strychnos icaja.

A reinvestigation of Strychnos icaja roots has resulted in the isolation of two tertiary quasi-symmetric bisindole alkaloids named strychnogucines A (1) and B (2). Their structures were identified by means of spectroscopic data interpretation. Compound 2 was highly active in vitro and compound 1 moderately active against four strains of Plasmodium falciparum. Strychnogucine B (2) was more active against a chloroquine-resistant strain than against a chloroquine-sensitive one (best CI(50), 80 nM against the W2 strain). In addition, this compound showed a selective antiplasmodial activity with 25-180 times greater toxicity toward P. falciparum, relative to cultured human cancer cells (KB) or human fibroblasts (WI38).

New antimalarial and cytotoxic sungucine derivatives from Strychnos icaja roots.

Reinvestigation of Strychnos icaja Baillon resulted in the isolation of vomicine, isostrychnine and of three new sungucine derivatives, named isosungucine (8), 18-hydroxy-sungucine (9) and 18-hydroxy-isosungucine (10). They were identified by detailed spectroscopic methods. The complete 1H- and 13C-NMR study of sungucine was also realized. Some of these compounds were highly active against Plasmodium falciparum in vitro and more particularly against the chloroquine-resistant strain. Compound 10 showed a selective antiplasmodial activity, with > 100-fold greater toxicity towards Plasmodium falciparum, relative to cultured human cancer cells (KB and HeLa lines) or fibroblasts (WI38).

Inhibition of croton oil-induced oedema in mice ear skin by capsular polysaccharides from cyanobacteria.

The anti-inflammatory properties of hydrophilic extracts of the capsular polymers of twelve cyanobacterial strains belonging to the genera Phormidium and Nostoc from marine and terrestrial habitats were tested topically on croton oil-induced oedema in mice ear skin. The screening program identified several strains as producers of anti-inflammatory products (up to 56% inhibition of the oedema). The inhibition response was dose-dependent. The application of trichloroacetic acid-treated extracts reduced the oedema by about 60%. On the other hand, one of the strains enhanced the inflammatory response. Analysis of five of the extracts showed the presence of neutral sugars (from 34.3% to 47.1%, w/w), uronic acids (from 7.1% to 26.7%, w/w) and proteins (from 30.1% to 57.0%, w/w) in the crude polymer. Rhamnose, fucose, arabinose, xylose, mannose, glucose, galactose, galacturonic acid and glucuronic acid were detected as well as sulphate groups (from 9.6% to 21.5%, w/w of sugars). The main components found were glucose and mannose.

Stimulation of topoisomerase II-mediated DNA cleavage by three DNA-intercalating plant alkaloids: cryptolepine, matadine, and serpentine.

Cryptolepine, matadine, and serpentine are three indoloquinoline alkaloids isolated from the roots of African plants: Cryptolepis sanguinolenta, Strychnos gossweileri, and Rauwolfia serpentina, respectively. For a long time, these alkaloids have been used in African folk medicine in the form of plant extracts for the treatment of multiple diseases, in particular as antimalarial drugs. To date, the molecular basis for their diverse biological effects remains poorly understood. To elucidate their mechanism of action, we studied their interaction with DNA and their effects on topoisomerase II. The strength and mode of binding to DNA of the three alkaloids were investigated by spectroscopy. The alkaloids bind tightly to DNA and behave as typical intercalating agents. All three compounds stabilize the topoisomerase II-DNA covalent complex and stimulate the cutting of DNA by topoisomerase II. The poisoning effect is more pronounced with cryptolepine than with matadine and serpentine, but none of the drugs exhibit a preference for cutting at a specific base. Cryptolepine which binds 10-fold more tightly to DNA than the two related alkaloids proves to be much more cytotoxic toward B16 melanoma cells than matadine and serpentine. The cellular consequences of the inhibition of topoisomerase II by cryptolepine were investigated using the HL60 leukemia cell line. The flow cytometry analysis shows that the drug alters the cell cycle distribution, but no sign of drug-induced apoptosis was detected when evaluating the internucleosomal fragmentation of DNA in cells. Cryptolepine-treated cells probably die via necrosis rather than via apoptosis. The results provide evidence that DNA and topoisomerase II are the primary targets of cryptolepine, matadine, and serpentine.

The plant alkaloid usambarensine intercalates into DNA and induces apoptosis in human HL60 leukemia cells.

Usambarensine is a plant alkaloid isolated from the roots of Strychnos usambarensis collected in Central Africa. This bis-indole compound displays potent antiamoebic activities and shows antigardial, antimalarial and cytotoxic effects. Usambarensine is highly toxic to B16 melanoma cells and inhibits the growth of leukemia and carcinoma cells. To date, the molecular basis for its diverse biological effects remains totally unknown. However, its capacity to inhibit nucleic acids synthesis in melanoma cells, on the one hand, and its structural analogy with DNA-binding pyridoindole plant alkaloids recently studied (cryptolepine and matadine), on the other hand, suggested that usambarensine could also bind to DNA. Consequently, we studied the strength and mode of binding to DNA of usambarensine by means of absorption, circular and linear dichroism. The results of the optical measurements indicate that the alkaloid effectively binds to DNA and behaves as a typical intercalating agent. Biochemical experiments indicated that, in contrast to cryptolepine and matadine, usambarensine does not interfere with the catalytic activity of topoisomerase II. Human HL60 leukemia cells were used to assess the cytotoxicity of the alkaloid and its effect on the cell cycle. Usambarensine treatment is associated with a loss of cells in the G1 phase accompanied with a large increase in the sub-G1 region which is characteristic of apoptotic cells. The DNA of usambarensine-treated cells was severely fragmented and the proteolytic activity of DEVD-caspases is enhanced. Usambarensine is thus characterized as DNA intercalator inducing apoptosis in leukemia cells.

The DNA intercalating alkaloid cryptolepine interferes with topoisomerase II and inhibits primarily DNA synthesis in B16 melanoma cells.

Cryptolepine hydrochloride is an indoloquinoline alkaloid isolated from the roots of Cryptolepis sanguinolenta. It is characterized by a multiplicity of host-mediated biological activities, including antibacterial, antiviral, and antimalarial properties. To date, the molecular basis for its diverse biological effects remains largely uncertain. Several lines of evidence strongly suggest that DNA might correspond to its principal cellular target. Consequently, we studied the strength and mode of binding to DNA of cryptolepine by means of absorption, fluorescence, circular, and linear dichroism, as well as by a relaxation assay using DNA topoisomerases. The results of various optical and gel electrophoresis techniques converge to reveal that the alkaloid binds tightly to DNA and behaves as a typical intercalating agent. In DNAase I footprinting experiments it was found that the drug interacts preferentially with GC-rich sequences and discriminates against homo-oligomeric runs of A and T. This study has also led to the discovery that cryptolepine is a potent topoisomerase II inhibitor and a promising antitumor agent. It stabilizes topoisomerase II-DNA covalent complexes and stimulates the cutting of DNA at a subset of preexisting topoisomerase II cleavage sites. Taking advantage of the fluorescence of the indoloquinoline chromophore, fluorescence microscopy was used to map cellular uptake of the drug. Cryptolepine easily crosses the cell membranes and accumulates selectively into the nuclei rather than in the cytoplasm of B16 melanoma cells. Quantitative analyses of DNA in cells after Feulgen reaction and image cytometry reveal that the drug blocks the cell cycle in G2/M phases. It is also shown that the alkaloid is more potent at inhibiting DNA synthesis rather than RNA and protein synthesis. Altogether, the results provide direct evidence that DNA is the primary target of cryptolepine and suggest that this alkaloid is a valid candidate for the development of tumor active compounds.

Characterization of non pigmented B16 melanoma cell-derived cytotoxic factors.

We analyzed and tried to characterize substance(s) responsible for cytotoxic activities detected in culture media conditioned by non pigmented B16 melanoma cells (NPB16). The different cytological tests used showed that ultrafiltrated conditioned media (CM U1 fraction) contained several cytotoxic factors with a Mw lower than 1000 Da. These factors seemed to act either directly or indirectly on cell membranes, mitochondria, on the cell cycle and on protein and DNA synthesis. A cytotoxic activity could be found even after high dilution of CM U1. These cytotoxic factors were rapidly released by B16 cells in culture, independently of cell confluence. Their activities in the treated cells were also very fast and the cytotoxic effects were irreversible after only a few hours of treatment. These factors were not intermediate products during melanogenesis, neither polyamines, nor proteases. At least one of them seemed to be a small acidic and basic stable peptide without disulfide bounds but not heat stable. The synthesis of at least one of these cytotoxic factors was inhibited by cycloheximide and the cytotoxic activity was partially destroyed by pronase and trypsin, but not by pepsin. The cytotoxicity was not modified by copper complexants or free radical inhibitors (bovine serum albumin (BSA), tyrosine, superoxyde dismutase (SOD), catalase, vitamin E). Furthermore the levels of glutathione peroxydase activity and reduced glutathione did not change after treatment by CM U1 as compared to controls.

In vitro cytotoxic activity of two potential anticancer drugs isolated from Strychnos: strychnopentamine and usambarensine.

The cytotoxicity and the selective antiprotozoal activity of some Strychnos alkaloids, namely strychnopentamine (SP) and usambarensine (US) (7) led us to analyze and compare their effects with emetine (EM) by using mouse B16 melanoma cells cultivated in vitro. We observed by cytological analysis and proliferation rate studies that these substances induce analogous cytotoxic effects in B16 cells, but at different concentrations i.e. formation of lamellar bodies in the cytoplasm, the which contain pre-melanosomes in the case of SP and US, vacuoles and blebs. At concentrations near their respective IC50, SP and US, but not EM, decreased colony formation. We showed by incorporation of labelled precursors that SP and US first inhibit RNA synthesis while EM initially acts on protein synthesis. These alkaloids increased melanin synthesis. Furthermore, only EM and SP caused hemolysis of sheep red blood corpuscles. This could explain why the rate of antiplasmodial activity is higher for SP and EM.

Effects of alpha-hederin, a saponin extracted from Hedera helix, on cells cultured in vitro.

In this work, we have analysed the effects of alpha-hederin, a monodesmosidic triterpenoid saponin isolated from Hedera helix, on mouse B16 melanoma cells and non-cancer mouse 3T3 fibroblasts cultured in vitro. Our results indicate that, in a serum-free medium, alpha-hederin is cytotoxic and inhibits proliferation in both cell lines at rather low concentrations (< 5 micrograms/ml) after only 8 hours of treatment. Its cytotoxicity decreases in the presence of serum in which BSA seems to be able to bind the saponin. alpha-Hederin also induces vacuolization of the cytoplasm and membrane alterations leading to cell death.

Strychnopentamine, a potential anticancer agent.

We analysed the effects of strychnopentamine, an alkaloid isolated from Strychnos usambarensis, on an Ehrlich ascites tumor growing in the mouse after inoculation. Four subcutaneous injections of 1.5 mg strychnopentamine (1 per day) induce a significant decrease of the number of tumor cells and a significant increase of the survival of the treated mice. Observed side effects are partial haemolysis and some liver damage.

Effects of strychnopentamine on cells cultured in vitro.

This paper describes the powerful cytotoxic action exerted by strychnopentamine (SP), a dimeric indole alkaloid extracted from Strychnos usambarensis Gilg, on B16 melanoma cells and on non-cancer human fibroblasts cultured in vitro. SP strongly inhibits cell proliferation and induces cell death at a relatively low concentration (less than 1 microgram/ml) after 72 h of treatment in the two lines. Incorporation of [3H]thymidine and [3H]leucine by B16 cells significantly decreases after only 1 h of treatment at 0.5 microgram/ml. SP induces the formation of dense lamellar bodies and vacuolization in the cytoplasm, intense blebbing at the cell surface and various cytological alterations leading to cell death.

Why further investigation of brazilian Strychnos?

During the past fifteen years, African Strychnos species have been pharmacologically and chemically screened by some European Universities, sometimes with the collaboration of some African Institutes. Strychnos usambarensis will be discussed here because of our interest in it, after the discovery of its use in the preparation arrow poison with curarizing activity.The European studies have show thah the African species of Strychnoschemically are very similar to certain South-American species. Indeed, the occurrence in the African species of curarizing quaternary bases, and in the American species of tetanizing tertiary bases, such as akagerine, emphasizes the unity of genus Strychnos in terms of it alkaloids. Many biological activities (antmicrobial, hypotensive, smasmolytic, amoebicial...) have been detected.Finally, the antimitotic properties of some alkaloids will be presented. Besides know antitumor drugs, e.g. alstonine and ellipticine, African species possess new antimitotic products, e.g. strychnopentamine present in Strychnos usambarensis.Because of these findings, the main reasons why further investigate Brazilian Strychnos species are the lack of knowledge about:- the distribution on the species in the southern part of the great Amazon Basin.Accordin to Krukoff, it is possible that new species may be found there.- the distribution of alkaloids in many of know species. Indeed, there is little information on the variation in compposition of the alkaloidal mixtures in the different parts of the plants.- the biological of alkaloids. In the past, interest has been focused too narrowly on possible curarizing properties.Why should Brazilian species not cantain alkaloids with properties similar to those found in the African species?