Isolation and biological activity of compounds from Garcinia preussii.

CONTEXT: Plants of the genus Garcinia (Clusiaceae) are traditionally used to relieve stomachaches, toothaches, and as a chew stick. OBJECTIVE: In order to determine which compounds were responsible for these activities, a phytochemical investigation of the fruits and leaves of Garcinia preussii Engl. was pursued. MATERIALS AND METHODS: Plants were extracted by solvents of various polarities. Compounds isolation was then carried out using chromatography methods (medium- and high-pressure liquid chromatography, open column and thin-layer chromatography). The isolated compounds were identified and characterized by using 1D and 2D NMR spectroscopies. The antioxidant activity was evaluated using DPPH(•), ABTS(•-), ALP, and ORAC assays. The antimicrobial activity was assayed against Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, and Enterococcus faecalis by determining the minimum inhibitory concentration (MIC) value. The cytotoxic activity of most of the isolated compounds was evaluated on a small panel of human cancer cell lines (DU145, HeLa, HT-29, and A431) using the XTT method. RESULTS: The phytochemical investigation of G. preussii led to the isolation of eight known compounds, six benzophenones and two flavonoids. These compounds were tested for their biological activities. 1, 2, 3, 4, 7 and 8 demonstrated a high free radical scavenging activity with ER50 ranging from 0.1 to 0.7. The antimicrobial activity was shown only against Gram-positive bacteria for 1, 4, and 5. A moderate cytotoxic activity with IC50 ranging from 7 to 50 µM was observed, except for 6 which was not active. CONCLUSION: These results appear to support some of the properties reported for Garcinia species.

Peltogynoids and 2-phenoxychromones from Peltophorum pterocarpum and evaluation of their estrogenic activity.

Phytochemical investigation of the dichloromethane extract of the leaves of Peltophorum pterocarpum, a tropical ornamental tree, led to the isolation of twelve compounds (1-12). One new derivative of peltogynoid ophioglonin (1) and a new 2-phenoxychromone (2) with its 3'-O-ß-D-glucoside derivative (3) are described here for the first time. In addition, nine flavonoid derivatives, including peltogynoid ophioglonin (4), were isolated for the first time from this plant. The structures were determined by spectroscopic and chemical methods. Evaluation of the estrogenic activities of 1, 2, and 4 using different model cell systems revealed that 4 was estrogenic and that 2 was largely inactive. Interestingly, 1 was unable to stimulate the proliferation of breast and endometrial cancer cells but exhibited substantial estrogen receptor α-mediated activation of gene expression. This observation indicates that 1 can be further evaluated for its cancer chemopreventive potential.

Preussianone, a new flavanone-chromone biflavonoid from Garcinia preussii Engl.

A new flavanone-chromone biflavonoid, preussianone (1), has been isolated from the leaves of Garcinia preussii, along with four known biflavonoids. The absolute stereostructures were elucidated by chemical, spectroscopic, and chiroptical methods. The biological properties of the new biflavonoid against several bacterial strains were evaluated.

Schizanthines N, O, and P, tropane alkaloids from the aerial parts of Schizanthus tricolor.

Three tropane alkaloids, named schizanthines N, O, and P (1-3), have been isolated from the crude alkaloid extract of the endemic Chilean plant Schizanthus tricolor. On the basis of extensive NMR studies and MS fragmentation analysis, their structures were determined to be 3α-(E)-4-hydroxysenecioyloxy-6ß-angeloyloxytropane (1), 3α-(E)-4-hydroxysenecioyloxy-6ß-senecioyloxytropane (2), and 3α-mesaconyloxy-6ß-senecioyloxytropane (3). Compounds 1 and 2 are the first isomeric alkaloids in the tropane series possessing a hydroxysenecioyl substituent as an esterifying moiety.

Structure elucidation and NMR assignments of two new triterpenoids from the stems of Paragonia pyramidata (Bignoniaceae).

Phytochemical investigation of dichloromethane (DCM) extract from the stems of Paragonia pyramidata var. pyramidata L. Rich. (Bur.) resulted in the isolation and characterization of two new triterpenoids 3ß,19ß-dihydroxylup-12, 20(29)-diene-28-oic acid (1) and 3ß,19ß-dihydroxylup-12-en-28-oic acid (2), three known triterpenoids lupeol (3), spinosic acid A (4) and oleanolic acid (5), together with four known steroids (20R)-22E-24-ethylcholesta-4,22-dien-3-one (6), (20R)-24-ethylcholest-4-en-3-one (7), stigmasterol (8) and ß-sitosterol (9). HREIMS, GC-MS and NMR experiments including HSQC, HMBC, COSY and NOESY were used for the determination of the structures and NMR spectral assignments. This is the first report about the chemical constituents for this plant.

Metabolic profiling of Rhodiola rosea rhizomes by ¹H NMR spectroscopy.

INTRODUCTION: Rhodiola rosea is a broadly used medicinal plant with largely unexplored natural variability in secondary metabolite levels. OBJECTIVE: The aim of this work was to develop a non-target procedure for ¹H NMR spectroscopic fingerprinting of rhizome extracts for pattern recognition analysis and identification of secondary metabolites responsible for differences in sample composition. To achieve this, plants from three different geographic areas (Swiss Alps, Finland, and Altai region in Siberia) were investigated. RESULTS: A sample preparation procedure was developed in order to remove polymeric polyphenols as the ¹H NMR analysis of low-molecular-weight metabolites was hampered by the presence of tannins. Principal component analysis disclosed tight clustering of samples according to population. PCA models based on the aromatic region of the spectra showed that the first two components reflected changes in the content of salidroside and rosavin, respectively, the rosavin content being negatively correlated to that of rhodiocyanoside A and minor aromatics. Score plots and non-parametric variance tests demonstrated population-dependent changes according to harvest time. Data consistency was assessed using score plots and box-and-whisker graphs. In addition, a procedure for presenting loadings of PCA models based on bucketed data as high-resolution plots, which are reminiscent of real ¹H NMR spectra and help to identify latent biomarkers, is presented. CONCLUSION: This study demonstrated the usefulness of the established procedure for multivariate non-target ¹H NMR metabolic profiling of Rhodiola rosea.

Anti-inflammatory, antioxidant and antifungal activity of Chuquiraga spinosa.

CONTEXT: Stem and leaves infusion of Chuquiraga spinosa (R&P) Don. (Asteraceae) is used in the Peruvian traditional medicine for its anti-inflammatory properties and for the treatment of vaginal infections. OBJECTIVE: This study evaluated the antioxidant, anti-inflammatory and antifungal activities of C. spinosa for the first time. MATERIALS AND METHODS: Extracts of methanol, 50% methanol and water were obtained from C. spinosa aerial parts. Antioxidant activity of the extracts was evaluated (DPPH˙, ABTS˙(+) and superoxide radical-scavenging activity). The correlation between these results and total polyphenolic content was determined by Pearson's Correlation Coefficient. Anti-inflammatory activity of 50% methanol extract was evaluated with the rat model of carrageenan-induced acute inflammation and mouse model of TPA-induced acute inflammation. The antifungal activity of the extracts against Cladosporium cucumerinum and Candida albicans was studied by direct bioautography, and antifungal activity against phytopathogenic fungi was performed by culture in potato dextrose agar plates. RESULTS: All the extracts showed high antioxidant activity, and there was correlation between the activity and total polyphenolic compounds. As 50% methanol extract was administered orally, the paw edema in rats was reduced significantly (52.5%). This extract, by topical administration, produced a reduction of 88.07% of the edema TPA-induced in ear of mice. The aqueous and 50% methanol extracts were active against C. albicans (minimum inhibitory concentration of 2.5 and 6.25 µg, respectively). The aqueous extract showed antifungal activity against C. cucumerinum (MIC: 2.5 µg). DISCUSSION AND CONCLUSION: Preliminary phytochemical screening and the analysis of the three extracts by high-performance liquid chromatography diode-array detection showed the majority compounds are flavonoids and phenolic acid derivatives. These compounds may be responsible of the radical-scavenging activity of these extracts as well as responsible of anti-inflammatory effect in vivo of 50% methanol extract. Several authors have demonstrated the antioxidant and anti-inflammatory properties of some flavonoids and phenolic acids. The antifungal activity of the extracts obtained from aerial parts of C. spinosa has been investigated here for the first time. Other studies are necessary to determine the mechanism of action and to identify the bioactive compounds of this plant.

Isomeric tropane alkaloids from the aerial parts of Schizanthus tricolor.

Investigation of the aerial parts of Schizanthus tricolor yielded seven isomeric tropane alkaloids: 3alpha-(1-methylitaconyl)-6beta-senecioyloxytropane (1), 3alpha-(1-methylitaconyl)-6beta-angeloyloxytropane (2), 3alpha-(1-methylmesaconyl)-6beta-senecioyloxytropane (3), 3alpha-(1-methylmesaconyl)-6beta-angeloyloxytropane (4), 3alpha-(1-methylmesaconyl)-6beta-tigloyloxytropane (5), 3alpha-(1-methylcitraconyl)-6beta-senecioyloxytropane (6), and 3alpha-(1-methylcitraconyl)-6beta-angeloyloxytropane (7). Their structures were established by NMR including (1)H, (13)C NMR, HSQC, HMBC, COSY, and NOESY experiments, UV, IR, and mass spectrometry. Compounds 1, 6, and 7 are new to the literature. Alkaloids 1, 3, 4, and 5 and a mixture of 3, 4, and 5 were evaluated for in vitro antiplasmodial and cytotoxic activity. Compounds 1, 4, and 5 showed marginal inhibition of Plasmodium falciparum strain K1 with IC(50) values of 22.8, 24.8, and 36.0 microM and displayed no cytotoxicity on MRC-65 cells (CC(50) > 64 microM). Alkaloid 3 was inactive (IC(50) 63.5 microM). The alkaloid mixture exhibited slightly higher activity (IC(50) 17.0 microM) than the pure compounds, indicating some synergy between the different isomers.

Antimalarial activity of extract and norbergenin derivatives from the stem bark of Diospyros sanza-minika A. Chevalier (Ebenaceae).

The methanol extract from the stem bark of Diospyros sanza-minika as well as five norbergenin derivatives isolated from this crude extract were evaluated for their in vitro activity against Plasmodium falciparum K1 and cytotoxicity on MRC-5 cells. 4-O-(3'-methylgalloyl)norbergenin was found to be the most potent compound (IC(50) 0.6 µg/mL; CC(50) 24.7 µg/mL), followed by 4-O-galloylnorbergenin (IC(50) 3.9 µg/mL; CC(50) > 64 µg/mL) and 11-O-p-hydroxy-benzoyl-norbergenin (IC(50) 4.9 µg/mL; CC(50) > 64 µg/mL). Norbergenin and 4-O-syringoylnorbergenin were inactive (IC(50) > 32 µg/mL; CC(50) > 64 µg/mL). The antimalarial activity of the pure constituents and of the methanol extract from the stem bark of Diospyros sanza-minika is reported for the first time. The results provide interesting baseline information for the potential use of the crude extract well as some of the isolated compounds in the search for novel antimalarial compounds.

High-precision heteronuclear 2D NMR experiments using 10-ppm spectral window to resolve carbon overlap.

The acquisition of a complementary heteronuclear 2D NMR experiment with 10-ppm carbon window allows chemists to improve by a factor 20-25 the spectral resolution and determine carbon chemical shifts with five figures from 2D spectra.

Antioxidant phenylethanoid glycosides and a neolignan from Jacaranda caucana.

Extracts from several plants of the family Bignoniaceae from Panama were submitted to a rapid DPPH TLC test for the detection of radical-scavenging activity. The MeOH extract of the stems of Jacaranda caucana, a tree that grows from Costa Rica to Colombia, was selected due to its interesting activity and the lack of phytochemical studies on the polar extract. This extract was partitioned between ethyl acetate, butanol, and water. The EtOAc fraction afforded two new phenylethanoid glycosides (1, 2), along with protocatechuic acid, acteoside, and jionoside D. Further purifications yielded isoacteoside and martynoside. The BuOH fraction afforded a new rhamnosyl derivative of sisymbrifolin (8), a neolignan. The structures were determined by means of spectrometric methods, including 1D and 2D NMR experiments and MS analysis.

Rapid separation of three glucosylated resveratrol analogues from the invasive plant Polygonum cuspidatum by high-speed countercurrent chromatography.

Three glucosylated resveratrol analogues (piceid, piceatannol glucoside, resveratroloside) were successfully isolated from the crude MeOH extract of the invasive plant species Polygonum cuspidatum by semi-preparative high-speed countercurrent chromatography with a two-phase solvent system composed of cyclohexane-ethyl acetate-methanol-water (1:5:1:5, v/v/v/v). Piceid (23 mg), resveratroloside (17 mg), piceatannol glucoside (15 mg) of purities over 80% were isolated from 500 mg crude MeOH extract in one step. Subsequent passage over a SPE column was used to quickly bring their purities to over 90%. The purities were determined by HPLC analysis and their structures were elucidated by proton nuclear magnetic resonance ((1)H-NMR), HMBC, ESI-MS and HR-MS.

Natural product analysis over the last decades.

Progress in natural product chemistry has always been strongly linked to innovations in analytical technology. The characterisation of metabolites in complex mixtures requires sophisticated techniques, which should provide good sensitivity and selectivity as well as structural information on the constituents of interest. This review outlines the most important chromatographic and spectral techniques which have been introduced in the field of natural products. Although there has been a very rapid evolution of methods over the last 50 years, the introduction of high-throughput screening programmes require even more efficient and sensitive methodologies which yield adequate on-line information for metabolite structure determination.

Ultra-performance liquid chromatography/time-of-flight mass spectrometry as a chemotaxonomic tool for the analysis of Gentianaceae species.

INTRODUCTION: The segregation between the genera Gentiana and Gentianella among the Gentianaceae family is poorly defined. In order to clarify the classification of these genera, some researchers have tried to incorporate data about the chemical constitution, but this has not yet been achieved in a comprehensive way. OBJECTIVE: To develop a fast and reproducible analytical method for the observation of characteristic fingerprints of secondary metabolites of each genus. METHODOLOGY: Seven species were investigated, three Gentianella and four Gentiana selected for their close taxonomic links within each genus. Ten xanthones previously isolated from one of these species were used as chemotaxonomic markers. A UPLC/ESI-TOF-MS method was developed to analyse the methanolic extracts. RESULTS: The UPLC/TOF-MS provided clear metabolic fingerprints and elemental composition of the compounds. The profiles of the three Gentianella species were strikingly similar. On the contrary, metabolic profiles of Gentiana species were very different from the Gentianella chromatograms and also from each other. Several compounds were unique to each genus and therefore could be used as biomarkers. CONCLUSION: UPLC/TOF-MS can be applied as a chemotaxonomic tool for the rapid screening of Gentianaceae species and for the distinction between closely related taxa from the Gentianaceae family.

A TLC bioautographic method for the detection of alpha- and beta-glucosidase inhibitors in plant extracts.

INTRODUCTION: Bioautographic assays using TLC play an important role in the search for active compounds from plants. A TLC assay has previously been established for the detection of beta-glucosidase inhibitors but not for alpha-glucosidase. Nonetheless, alpha-glucosidase inhibition is an important target for therapeutic agents against of type 2 diabetes and anti-viral infections. OBJECTIVE: To develop a TLC bioautographic method to detect alpha- and beta-glucosidase inhibitors in plant extracts. METHODOLOGY: The enzymes alpha- and beta-d-glucosidase were dissolved in sodium acetate buffer. After migration of the samples, the TLC plate was sprayed with enzyme solution and incubated at room temperature for 60 min in the case of alpha-d-glucosidase, and 37 degrees C for 20 min in the case of beta-d-glucosidase. For detection of the active enzyme, solutions of 2-naphthyl-alpha-D-glucopyranoside or 2-naphthyl-beta-D-glucopyranoside and Fast Blue Salt were mixed at a ratio of 1 : 1 (for alpha-d-glucosidase) or 1 : 4 (for beta-d-glucosidase) and sprayed onto the plate to give a purple background colouration after 2-5 min. RESULTS: Enzyme inhibitors were visualised as white spots on the TLC plates. Conduritol B epoxide inhibited alpha-d-glucosidase and beta-d-glucosidase down to 0.1 microg. Methanol extracts of Tussilago farfara and Urtica dioica after migration on TLC gave enzymatic inhibition when applied in amounts of 100 microg for alpha-glucosidase and 50 microg for beta-glucosidase. CONCLUSION: The screening test was able to detect inhibition of alpha- and beta-glucosidases by pure reference substances and by compounds present in complex matrices, such as plant extracts.

Antiproliferative effects of withanolides from Withania adpressa.

Extracts of Withania adpressa Coss. (Solanaceae), a medicinal plant endemic to Moroccan Sahara, were tested for their cytotoxicity towards a panel of cancer cell lines (Hep2, HT29, RD, Vero and MDCK), using the (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) [MTT assay, Sigma-Aldrich]. The bioassay-guided fractionation of this plant extracts results a novel withanolide 14alpha,15alpha,17beta ,20beta-tetrahydroxy-1-oxo-(22R)-witha-2,5,24-trienolide and the already identified withanolides F and J extract, semi-purified fractions and pure compounds exhibits potent cytotoxicity against human cancer cell lines tested, in dose-dependent manner. Morphological features of treated Hep2 cells with the novel withanolide and characteristic DNA fragmentation revealed that the cytotoxicity was due to induction of apoptosis. Taken together, the results suggest that withanolides from W. adpressa Coss. hold potential as antiproliferative agents.

Antioxidant C-glucosylxanthones from the leaves of Arrabidaea patellifera.

Chemical investigation of the methanol extract from the leaves of Arrabidaea patellifera, a Bignoniaceae from Panama, afforded mangiferin, isomangiferin, and six new derivatives (3'-O-p-hydroxybenzoylmangiferin, 3'-O-trans-coumaroylmangiferin, 6'-O-trans-coumaroylmangiferin, 3'-O-trans-cinnamoylmangiferin, 3'-O-trans-caffeoylmangiferin, and 3'-O-benzoylmangiferin). All these compounds had antioxidant and radical-scavenging activities, and four of them were relatively active in vitro against Plasmodium falciparum. The structures were determined by spectrometric and chemical methods, including 1D and 2D NMR experiments and MS analysis.

In vitro screening assays to identify natural or synthetic acetylcholinesterase inhibitors: thin layer chromatography versus microplate methods.

Acetylcholinesterase inhibitors (AChEI) are currently still the best available pharmacotherapy for Alzheimer patients. Successful screening for new AChEI relies on effective and fast assays. Two colorimetric screening assays frequently used to search for new AChEI, namely a thin layer chromatography (TLC) assay with Fast Blue B salt as reagent and a 96-well plate assay based on Ellman's method, were compared. For the majority (83%) of the 138 test compounds of natural and synthetic origin, the results obtained with the two assays converged and both screening assays were considered suitable for the generation of new hits. Fifteen percent of investigated compounds were classified as active with the microplate assay but were shown to be inactive by TLC and about 2% were measured active by TLC but showed to be inactive with the microplate assay. These divergences were not due to the main differences between the experimental protocols of the two screening assays, namely the different colorimetric methods and pre-incubation of test compounds with acetylcholinesterase (AChE). They might be explained by the interaction of either AChE or test compounds with the silica of the TLC plates, resulting in an altered affinity of the enzyme for the compounds.