Liene Dhooghe

Synthesis and antiplasmodial activity of aminoalkylamino-substituted neocryptolepine derivatives.

A series of chloro- and aminoalkylamino-substituted neocryptolepine (5-methyl-5H-indolo[2,3-b]quinoline) derivatives were synthesized and evaluated as antiplasmodial agents. The evaluation also included cytotoxicity (MRC5 cells), inhibition of beta-hematin formation, and DNA interactions (DNA-methyl green assay). Introduction of aminoalkylamino chains increased the antiplasmodial activity of the neocryptolepine core substantially. The most efficient compounds showed antiplasmodial activities in the nanomolar range. N(1),N(1)-Diethyl-N(4)-(5-methyl-5H-indolo[2,3-b]quinolin-8-yl)pentane-1,4-diamine 11c showed an IC(50) of 0.01 microM and a selectivity index of 1800.

Antimalarial activity and toxicity evaluation of a quantified Nauclea pobeguinii extract.

AIM OF THE STUDY To evaluate the in vitro and in vivo antiplasmodial activity and toxicity of the aqueous and 80% EtOH extract of the stem bark of Nauclea pobeguinii (Pob. Ex. Pell.) Petit (Rubiaceae), a plant used in traditional medicine in DR Congo against malaria. MATERIALS AND METHODS The aqueous and 80% EtOH extract from N. pobeguinii stem bark, and its constituents (5S)-5-carboxystrictosidine, 19-O-methylangustoline, 3-O-beta-fucosylquinovic acid, 3-ketoquinovic acid and strictosamide, were evaluated for their in vitro activity against Plasmodium falciparum (chloroquine-sensitive Ghana-strain). The 80% EtOH extract, containing 5.6% strictosamide, was evaluated in vivo in the 4-day P. berghei mouse model, and in the P. yoelii N67 model. RESULTS All compounds were inactive or only moderately active in vitro. The aqueous and 80% EtOH extract displayed moderate in vitro activity with IC(50) values of 44 and 32 microg/mL, respectively, without apparent cytotoxicity on MRC-5 cells (CC50>64 microg/mL). Daily oral dosing of the 80% EtOH extract, at 300 mg/kg, resulted in 86% reduction of parasitaemia in the 4-day P. berghei mouse model, and 75% reduction in the P. yoelii N67 model. Prolonging oral dosing to 2 x 5 days, with an interval of 2 days, and oral administration of the 80% EtOH extract at 300 mg/kg induced 92% reduction of parasitaemia, and a mean survival time of 17 days. Strictosamide, the putative active constituent, may be metabolically activated in the gastrointestinal tract after oral administration. Levels of creatinin, urea, ALAT and ASAT remained unchanged after treatment. No acute toxicity was observed in mice after a single 2g/kg oral dose, nor after 4 weekly doses. No significant macroscopic or microscopic lesions were observed in heart, lung, spleen, kidney, liver, large intestine and brain. CONCLUSIONS These results can partly support and justify the use of N. pobeguinii in traditional medicine in the DR Congo for the treatment of uncomplicated malaria.

Structure–activity relationship of antiparasitic and cytotoxic indoloquinoline alkaloids, and their tricyclic and bicyclic analogues

Based on the indoloquinoline alkaloids cryptolepine (1), neocryptolepine (2), isocryptolepine (3) and isoneocryptolepine (4), used as lead compounds for new antimalarial agents, a series of tricyclic and bicyclic analogues, including carbolines, azaindoles, pyrroloquinolines and pyrroloisoquinolines was synthesized and biologically evaluated. None of the bicyclic compounds was significantly active against the chloroquine-resistant strain Plasmodium falciparum K1, in contrast to the tricyclic derivatives. The tricyclic compound 2-methyl-2H-pyrido[3,4-b]indole (9), or 2-methyl-beta-carboline, showed the best in vitro activity, with an IC(50) value of 0.45 microM against P. falciparum K1, without apparent cytotoxicity against L6 cells (SI>1000). However, this compound was not active in the Plasmodium berghei mouse model. Structure-activity relationships are discussed and compared with related naturally occurring compounds.

Development and validation of an HPLC-method for the determination of alkaloids in the stem bark extract of Nauclea pobeguinii

A new method was developed and validated for the quantification of strictosamide in the extract of the stem bark of Nauclea pobeguinii. This plant belongs to the Rubiaceae family and is widely used in the African traditional medicine against malaria and malaria-like symptoms. Alkaloids are suspected to be responsible for the antimalarial activity. One of these alkaloids is strictosamide, already reported to be the major constituent in the root bark of this plant. Because strictosamide was not commercially available another alkaloid, ajmalicine HCl, with comparable properties was used as a secondary standard. The samples of the dried 80% ethanol extract from the stem bark of N. pobeguinii were purified on C(18) solid phase extraction cartridges and analysed using HPLC-UV. The strictosamide used for the validation of the correction factor for response was isolated and purified by means of preparative HPLC and TLC. Although the relative standard deviation (R.S.D.) of 2.6% was still acceptable, the response factor was determined for every analysis based on the ratio of the peak area of strictosamide compared to the peak area of ajmalicine HCl in a concentration of 0.01 mg/ml. The precision of the method according to the time and the concentration, had a R.S.D. value of 2.2% and 2.6%, respectively. The recovery of the method was 92.2% (R.S.D. of 9.4%) which was acceptable. The method has been proven to be suitable for the determination of alkaloids in the extract of the stem bark of N. pobeguinii, according to the ICH guidelines on the validation of analytical methods.

Herbal Medicines and Infectious Diseases: Characterization by LC‑SPE‑NMR of Some Medicinal Plant Extracts Used against Malaria

The extracts of two medicinal plants used in traditionalmedicine against malariawere characterized by means of an LC‑SPE‑NMR and LC‑MS platform. The structure of a series of major constituents from Bafodeya benna, as well as minor constituents from Ormocarpum kirkii, was determined. Bafodeya benna was found to contain (2R,3R)-taxifolin-3-O-α-L-rhamnoside or astilbin, and its isomers neoastilbin, neoisoastilbin, and isoastilbin, as well as quercetin-3-O-α-L-rhamnoside. From Ormocarpum kirkii, a series of known flavonoids and biflavonoids was obtained, as well as three new compounds, i.e., 7,7′′-di-O-β-D-glucosyl-(−)-chamaejasmin, 7-O-β-D-glucosyl-(I-3,II-3)-biliquiritigenin, and isovitexin-(I-3,II-3)-naringenin. The isolated constituents may explain, at least in part, the traditional use against malaria. LC‑SPE‑NMR, in combination with LC‑MS, is a powerful tool for the fast characterization of plant extracts, in order to define priorities at an early stage of a fractionation procedure. In addition, herbal medicinal products can completely be characterized, both with regard to their major as well as their minor constituents.

Quantification of xanthohumol, isoxanthohumol, 8-prenylnaringenin, and 6-prenylnaringenin in hop extracts and derived capsules using secondary standards

Hop is a well-known and already frequently used estrogenic phytotherapeutic, containing the interesting prenylflavonoids, xanthohumol (XN), isoxanthohumol (IXN), 8- and 6-prenylnaringenin (8-PN and 6-PN). Since the use of secondary standards can form a solution whenever the determination is required of certain components, not commercially available or too expensive, it was decided to develop an accessible HPLC-DAD method for the determination of these prenylflavonoids. The amounts were determined in hop extract and capsules, using quercetin and naringenin as secondary standards. After optimization of the sample preparation and HPLC conditions, the analysis was validated according to the ICH guidelines. The response function of XN, 8-PN, quercetin and naringenin showed a linear relationship. For the determination of XN, a calibration line of at least three concentrations of quercetin has to be constructed. The correction factors for XN (quercetin) and for 8-PN (naringenin) were validated and determined to be 0.583 for XN, and 1.296 for IXN, 8-PN and 6-PN. The intermediate precision was investigated and it could be concluded that the standard deviation of the method was equal considering time and concentration (RSD of 2.5-5%). By means of a recovery experiment, it was proven that the method is accurate (recoveries of 96.1-100.1%). Additionally, by analysing preparations containing hop extracts on the Belgian market, it was shown that the method is suitable for its use, namely the determination of XN, IXN, 8-PN and 6-PN in hop extract and capsules, using quercetin and naringenin as secondary standards.

Rapid isolation and identification of minor natural products by LC–MS, LC–SPE–NMR and ECD: Isoflavanones, biflavanones and bisdihydrocoumarins from Ormocarpum kirkii

The combination of the hyphenated techniques LC-MS and LC-SPE-NMR constitutes a powerful platform for the rapid isolation and identification of minor components from natural sources. Electronic circular dichroism (ECD) is a useful tool to determine the absolute configuration of small quantities of chiral molecules. In order to search for minor constituents present in an Ormocarpum kirkii extract, these techniques were applied for the separation and structure elucidation of a series of isoflavanones, biflavanones and biscoumarins. After optimization of chromatographic conditions and subsequent isolation, MS and 1D and 2D NMR data were collected. Experimental and calculated ECD spectra were used in conjunction with NMR data to confirm the absolute configuration of these compounds. Eight compounds were identified for the first time and six have been previously reported. The present approach offers a strategy for accelerating research on natural products.

The quantification of ellagic acid in the crude extract of Phyllanthus amarus Schum. & Thonn. (Euphorbiaceae).

INTRODUCTION Phyllanthus amarus Schum. & Thonn. (Euphorbiaceae), already well known for its antiviral, antihyperglycaemic and antihepatotoxic effects, is also investigated for its antimalarial activity. The major constituent of the crude extract of the whole plant was isolated and identified in this research to be ellagic acid, for which antiplasmodial activity already has been reported. OBJECTIVE Because of the potential of the plant and the interesting properties of ellagic acid, an analytical method can be useful for the standardisation of the extracts to allow further biological and pharmacological investigations. In order to obtain an easily performable and inexpensive method, an HPLC analysis was developed and validated. METHODOLOGY The samples were dissolved in DMSO, ultrasonicated for 15 min, and diluted with 50% methanol. Analysis was performed using water and methanol containing 0.06% TFA and the peaks were detected at 254 nm. RESULTS Ellagic acid showed a linear relationship in the range of 1.74-20.91 µg/mL and a single-point calibration was allowed. The method was shown to be precise with respect to time (RSD of 1.84%, 3 days, n = 6) and concentration (RSD of 2.54%, 3 levels, n = 6). The overall mean content of ellagic acid was 2.06%. A recovery experiment was performed and it showed an accuracy of 100.4%. CONCLUSION Based on the obtained results, it can be concluded that the newly developed method is suitable for its purpose, namely the determination of ellagic acid in the crude extract of P. amarus.