Reverse pharmacology for developing an anti-malarial phytomedicine. The example of Argemone mexicana.

Classical pharmacology has been the basis for the discovery of new chemical entities with therapeutic effects for decades. In natural product research, compounds are generally tested in vivo only after full in vitro characterization. However drug screening using this methodology is expensive, time-consuming and very often inefficient. Reverse pharmacology, also called bedside-to-bench, is a research approach based on the traditional knowledge and relates to reversing the classical laboratory to clinic pathway to a clinic to laboratory practice. It is a trans-disciplinary approach focused on traditional knowledge, experimental observations and clinical experiences. This paper is an overview of the reverse pharmacology approach applied to the decoction of Argemone mexicana, used as an antimalarial traditional medicine in Mali. A. mexicana appeared as the most effective traditional medicine for the treatment of uncomplicated falciparum malaria in Mali, and the clinical efficacy of the decoction was comparable to artesunate-amodiaquine as previously published. Four stages of the reverse pharmacology process will be described here with a special emphasis on the results for stage 4. Briefly, allocryptopine, protopine and berberine were isolated through bioguided fractionation, and had their identity confirmed by spectroscopic analysis. The three alkaloids showed antiparasitic activity in vitro, of which allocryptopine and protopine were selective towards Plasmodium falciparum. Furthermore, the amount of the three active alkaloids in the decoction was determined by quantitative NMR, and preliminary in vivo assays were conducted. On the basis of these results, the reverse pharmacology approach is discussed and further pharmacokinetic studies appear to be necessary in order to determine whether these alkaloids can be considered as phytochemical markers for quality control and standardization of an improved traditional medicine made with this plant.

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.

LC-DAD-MS-based metabolite profiling of three species of Justicia (Acanthaceae).

Olean-12-en-3ß-24 diol (A), auranamide (B), aurantiamide acetate (C), 2α,3ß-dihydroxy-olean-12-en-28-oic acid (D) and quindoline (E) were isolated from the dichloromethane (CH2Cl2) extract of the stems of Justicia secunda (Acanthaceae). Liquid chromatography with ultraviolet and mass spectrometric detection was used to acquire more knowledge of the chemical composition of this extract and to monitor variations in profiles of both the isolated and the other non-identified compounds in Justicia refractifolia and Justicia graciliflora. The compound classes, phenolic and olefinic amides, feruloyltyramine amides, 2,5-diaryl-3,4-dimethyltetrahydrofuranoid lignans, peptide alkaloids, phenylalanine derivatives, conjugated ynones, indolquinoline alkaloids, triterpenes and pigments, were tentatively identified based on the LC-DAD-APCI-MS analysis. The most frequently encountered compound among the species was auranamide while the distribution of quindoline was limited to J. secunda. Moreover, the acetylcholinesterase inhibitory activity of the isolated compounds was determined.

Anti-inflammatory Activity of the Invasive Neophyte Polygonum Cuspidatum Sieb. and Zucc. (Polygonaceae) and the Chemical Comparison of the Invasive and Native Varieties with regard to Resveratrol.

Polygonum cuspidatum Sieb. and Zucc. has been traditionally used as a member of many anti-inflammatory polyherbal formulations, but is now a widespread invasive neophyte in Europe and America. To discuss if the invasive variety is chemically identical to the native one in traditional medicine, the different constituents of the invasive variety compared to the native variety were isolated and their anti-inflammatory activity was tested. Resveratroloside and catechin-(4α→8)-catechin, the newly found constituents in the invasive variety, have similar nitric oxide (NO) inhibition potency as that of piceid (the major constituent of P. cuspidatum), but the newly found major constituent, i.e., piceatannol glucoside, showed no apparent effect. On the other hand, as a marker, the total content of resveratrol in the methanol root extract after glucosidase hydrolysis was measured and compared between the invasive and native varieties. The total content of resveratrol measured in the root extracts of the Swiss sample was about 2.5 times less than that of the Chinese one. This study brings attention to the point that when the invasive variety of P. cuspidatum is used in traditional medicine, the chemical difference should be kept in mind.

Antioxidant compounds from Algerian Convolvulus tricolor (Convolvulaceae) seed husks.

Seed husk extracts of Convolvulus tricolor L. (Convolvulaceae) afforded six compounds, identified for the first time from this plant: isorhamnetin 3-O-beta-D-galactopyranoside (1), isorhamnetin 3-O-beta-D-(6"-acetyl)-galactopyranoside (2), isorhamnetin 3-O-robinobioside (3), 3,4-di-O-caffeoylquinic acid (4), gentisic acid 5-O-glucoside (5), and scopoletin (6). Separation of compounds was carried out by CC and CPC. Structural elucidations were performed by HPLC-UV-DAD, HPLC-ESI/MS (negative mode) and NMR.

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.

Chemical constituents of Asplenium ruta-muraria L.

A phytochemical study of Asplenium ruta-muraria L. (Aspleniaceae) led to the isolation of a new caffeic acid glycoside, 2-O-caffeoyl-ß-D-fructofuranosyl-(2 → 1)-α-D-glucopyranoside and an (α, ß)-isomeric pair of 2E-caffeoyl-D-glucopyranoside, together with kaempferol-3-O-ß-D-[6-E-caffeoyl-ß-D-glucopyranosyl-(1 → 2)glucopyranoside]-7-O-ß-D-glucopyranoside, 1-O-caffeoyl glycoside, sucrose, diploptene and ß-sitosterol. Their structures were established by means of MS and capillary NMR techniques. Additionally, aromatase inhibitory activity of the extracts and phenolic compounds was evaluated.

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.

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.

Effects of an n-butanol extract from the stem of Tinospora crispa on blood pressure and heart rate in anesthetized rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Tinospora crispa has been used in folkloric medicine for control of blood pressure, as an antipyretic, for cooling down the body temperature and for maintaining good health. AIM OF THE STUDY: To investigate the effects and mechanisms of action of an n-butanol extract from the stems of Tinospora crispa (T. crispa extract) on blood pressure and heart rate in anesthetized rats. MATERIALS AND METHODS: Air-dried stems of T. crispa were extracted with water, followed by partitioned extract with chloroform, ethyl acetate, and finally by n-butanol. The n-butanol soluble part was evaporated under reduced pressure and lyophilization to obtain a crude dried powder (T. crispa extract). The effects and mechanisms of the T. crispa extract on blood pressure and heart rate were studied in anesthetized normal and reserpinized rats in vivo in the presence of different antagonists. RESULTS: T. crispa extract (1-100 mg/kg, i.v.) caused a decrease in mean arterial blood pressure (MAP) and this effect was inhibited by propranolol, phentolamine, atenolol and/or the ß(2)-antagonist ICI-118,551, but not by atropine or hexamethonium. In reserpinized rats, the T. crispa extract had a dual effect: reduction in hypotensive activity, followed by a small increase in blood pressure. The decrease in MAP in reserpinized rat was slightly potentiated by phentolamine, but inhibited by propranolol or ICI-118,551 only if atenolol and phentolamine were also present. The increase in MAP was potentiated by propranolol and ICI-118,551, but was inhibited by phentolamine. The T. crispa extract had a dual effect on heart rate in the normal rat: a small transient decrease, followed by an increase in heart rate. The positive chronotropic effect of T. crispa extract was inhibited by propranolol, phentolamine and atenolol, but not by ICI-118,551, atropine or hexamethonium. Reserpine potentiated the positive chronotropic effect of the T. crispa extract and this effect was inhibited by propranolol, atenolol and ICI-118,551, but not by phentolamine. CONCLUSIONS: From these results we suggest that T. crispa extract possesses at least three different cardiovascular-active components that act directly via (1) ß(2)-adrenergic receptors to cause a decrease in blood pressure, and ß(1)- and ß(2)-adrenergic receptors to cause an increase in heart rate, (2) α-adrenergic receptors to cause an increase in blood pressure and heart rate, and (3) a non-adrenergic and non-cholinergic pathway to cause a decrease in MAP and heart rate. These findings provide scientific support for the tradition of using this plant to modify the actions of the human cardiovascular system.

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.

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.

Hypotensive activity of an n-butanol extract and their purified compounds from leaves of Phyllanthus acidus (L.) Skeels in rats.

We aimed to investigate the effects, identify the active substances and establish the mechanisms involved in the hypotensive activity of an n-butanol extract from leaves of Phyllanthus acidus (PA extract). PA extract caused a decrease in blood pressure of anesthetized rats that was not modified by atropine or propranolol. PA extract caused a persistent dilatation of thoracic aortic rings preconstricted with either phenylephrine or KCl, and these effects were not modified by LNA or removal of the vascular endothelium. For phenylephrine-preconstricted aortic rings, the dilatory activity of the PA extract was not modified by atropine, propranolol or indomethacin. TEA, glybenclamide or ODQ significantly inhibited the dilatory activity of the PA extract on endothelium-denuded aortic rings. Nifedipine or a Ca(2+)-free medium depressed the aortic rings constrictor response to phenylephrine, and that was further augmented by the PA extract. Adenosine, 4-hydroxybenzoic acid, caffeic acid, hypogallic acid, and kaempferol were isolated from the PA extract. Each caused a decrease in blood pressure and dilatation of the aortic rings. LNA or removal of the endothelium reduced this activity. ODQ and TEA attenuated the vasodilatory activity of adenosine whereas glybenclamide and ODQ attenuated the effect of hypogallic acid. These results suggest that the hypotensive activities of the PA extract is likely the result of the direct action of these five compounds on the blood vessels by stimulating release of nitric oxide from the vascular endothelium, in part through stimulation of soluble guanylate cyclase, and opening of K(ATP) and K(Ca) channels in the vascular smooth muscle.

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.

Antioxidant and enzyme inhibition activities and chemical profiles of Polygonum sachalinensis F.Schmidt ex Maxim (Polygonaceae).

Polygonum sachalinensis is a widespread invasive plant in Europe. Chemical profiles of its different organs were studied by HPLC-UV-ESI/MS. Seven major constituents quercetin-3-O-beta-D-galactopyranoside, quercetin-3-O-arabinopyranoside, lapathoside D, N-trans-feruloyltyramine, lapathoside C, hydropiperoside, and vanicoside B were isolated and identified. The free radical-scavenging, alpha/beta-glucosidase, and acetylcholinesterase inhibitory activities of crude MeOH extracts and isolated compounds were studied. The structure-activity relationships were discussed. The chemical profiles revealed flavonoids and phenylpropanoids are the major compounds of all the organs of this plant. Quercetin-3-O-arabinopyranoside, lapathoside D, N-trans-feruloyltyramine, lapathoside C and hydropiperoside were isolated from this species for the first time. In the alpha-glucosidase bioassay, quercetin-3-O-beta-D-galactopyranoside, lapathoside D and N-trans-feruloyltyramine demonstrated stronger activities than the positive reference acarbose. The trend in scavenging power showed no relation to enzyme inhibition in the test models.

Two new tropane alkaloids from the bark of Erythroxylum vacciniifolium Mart. (Erythroxylaceae).

Two new tropane alkaloid N-oxides substituted by a methylpyrrole moiety were isolated from the bark of Erythroxylum vacciniifolium Mart. (Erythroxylaceae), a Brazilian indigenous plant, locally known as catuaba and used in traditional medicine as an aphrodisiac. The alkaloid structures were determined by a combination of high resolution mass spectrometry and multi-dimensional NMR spectroscopy.