UPLC-MS/MS-based molecular networking and NMR structural determination for the untargeted phytochemical characterization of the fruit of Crescentia cujete (Bignoniaceae).

The fruit pulp of Crescentia cujete is traditionally used in folk medicine for the treatment of a variety of respiratory conditions and gastrointestinal disorders. Due to the lack of a comprehensive phytochemical description of the fruit of this plant, its active compounds and rational quality control parameters have not yet been described. An untargeted metabolomics approach combining UPLC-MS/MS-based molecular networking with conventional isolation and NMR methods was carried out for the phytochemical profiling of the fruit pulp of Crescentia cujete. Sixty-six metabolites, including nine n-alkyl glycosides, twenty-three phenolic acid derivatives (such as cinnamoyl and benzoyl derivatives), fifteen flavonoids, four phenylethanoid derivatives and fifteen iridoid glycosides were identified at different levels of confirmation: eighteen confirmed structures (Level 1), six probable structures (Level 2) and forty two tentative candidates (Level 3). Among these, all four phenylethanoid derivatives were described for the first time within this species. In addition, 8-epi-eranthemoside, crescentiol A and crescentiol B were reported as three undescribed iridoid glucosides. The use of molecular networking has resulted in a detailed phytochemical overview of this species. This work provides a useful tool for further development and validation of appropriate analytical methods for routine quality control assessment of commercially available products containing the fruit of this species and further interpretation of their related pharmacological effects.

HPLC-DAD-SPE-NMR isolation of tetracyclic spiro-alkaloids with antiplasmodial activity from the seeds of Erythrina latissima.

Seven tetracyclic spiro-alkaloids, i.e. glucoerysodine (1), erysodine (2), epi-erythratidine (3), erysovine (4), erythratidine (5), erysotrine (6) and erythraline (7) were isolated from the seeds of Erythrina latissima by means of conventional separation methods and HPLC-DAD-SPE-NMR. Their structures were elucidated by spectroscopic means. This is the first report on the isolation of compounds 3, 5 and 6 from this plant. Antiplasmodial activity against the chloroquine-resistant strain Plasmodium falciparum K1 and cytotoxicity against MRC-5 cells (human fetal lung fibroblast cells) was assessed in vitro. Erysodine (2) and erysovine (4) showed moderate activity (IC50 6.53 µM and 4.05 µM, respectively), compared with the standard chloroquine (IC50 = 0.14 µM). No cytotoxicity was observed in a concentration up to 64.0 µM.

In vitro antigenotoxic activity, in silico ADME prediction and protective effects against aflatoxin B1 induced hepatotoxicity in rats of an Erythrina latissima stem bark extract.

Stem bark of Erythrina latissima E. Mey (Leguminosae) contains a wide range of prenylated flavonoids able to counteract the genotoxic properties of aflatoxin B1 (AFB1). Thus, the hypothesis was raised that E. latissima stem bark extracts (ELBE) may counteract the in vivo hepatotoxic effects of aflatoxins, contaminants in food and feed. An HPLC-DAD method was developed and validated to determine the level of flavonoid aglycones (11.82%) and glycosides (16.17%). ADME, pharmacokinetic and drug-likeness assessment of major flavonoids of ELBE, using the web tool SwissADME, showed good oral bioavailability. The protective effect of ELBE against AFB1 induced genotoxicity in the Vitotox assay after metabolic activation was confirmed (IC50 of 44.32 µg/ml), followed by evaluation of its inhibitory effect on hepatotoxicity in rats induced by the same agent. Male Wistar rats were orally treated with ELBE (20 mg/kg, 50 mg/kg and 100 mg/kg) or curcumin (500 mg/kg) combined with piperine (20 mg/kg) - positive control, for 8 days prior to AFB1 exposure (1 mg/kg). The ELBE group showed a decreased activity of ALP and γ-GT compared to the AFB1 group. Histopathological examination of the liver demonstrated ameliorative effects of ELBE. Thus, ELBE could have a protective effect against hepatotoxins such as AFB1.

Phytochemical characterization and comparative studies of four Cecropia species collected in Panama using multivariate data analysis.

Plant species of the genus Cecropia (Urticaceae) are used as traditional medicine in Latin-America, and are commercially available as food supplements. The aim of this study was to characterize and compare the phytochemical constituents of four Cecropia species collected in Panama. The structures of 11 compounds isolated from leaves of C. obtusifolia were elucidated based on high resolution mass spectrometry (HRMS) and nuclear magnetic resonance (NMR) spectroscopic analysis; the polyphenolic constituents of leaves of all four Cecropia species and commercial products were characterized using high performance liquid chromatography-diode array detection-quadrupole time of flight-tandem high resolution mass spectrometry (HPLC-DAD-QTOF). Forty-seven compounds were fully identified or tentatively characterized. Thirty-nine of these have not been previously reported for the species under investigation. Multivariate analysis revelead that C. obtusifolia and C. insignis are the most related species, while C. hispidissima is the most segregated one. Considering the importance of the description of novel chemical entities and the increasing interest and use of natural products, this study may be of great help for chemotaxonomic purposes, the interpretation of medicinal properties and for quality assessment of herbal supplements containing Cecropia leaves.

Ultrasound-assisted extraction optimization and validation of an HPLC-DAD method for the quantification of polyphenols in leaf extracts of Cecropia species.

Cecropia species are traditionally used in Latin American folk medicine and are available as food supplements with little information warranting their quality. The optimum conditions for the extraction of chlorogenic acid (CA), total flavonoids (TF) and flavonolignans (FL) from leaves of Cecropia species were determined using a fractional factorial design (FFD) and a central composite design (CCD). A reversed-phase high-performance liquid chromatographic method coupled to a diode array detector (HPLC-DAD) was validated for the quantification of CA, TF and FL, following the ICH guidelines. Quantitative and Principal Component Analysis (PCA) was also performed. The extraction-optimization methodology enabled us developing an appropriate extraction process with a time-efficient execution of experiments. The experimental values agreed with those predicted, thus indicating suitability of the proposed model. The validation parameters for all chemical markers of the quantification method were satisfactory. The results revealed that the method had excellent selectivity, linearity, precision (repeatability and intermediate precision were below than 2 and 5%, respectively) and accuracy (98-102%). The limits of detection and quantification were at nanogram per milliliter (ng/mL) level. In conclusion, the simultaneous quantification of chemical markers using the proposed method is an appropriate approach for species discrimination and quality evaluation of Cecropia sp.

Antimutagenic constituents from Monanthotaxis caffra (Sond.) Verdc.

OBJECTIVES: Monanthotaxis caffra (Sond.) Verdc. (Annonaceae) has been reported to possess antitumoural properties. Preliminary screening showed that the crude methanolic leaf extract had strong antimutagenic effects against aflatoxin B1 -induced mutagenicity. The aim of this study was to isolate and evaluate the antimutagenic properties of the active constituents from M. caffra. METHODS: Different chromatographic, spectroscopic and spectrometric techniques were used for the isolation and identification of the antimutagenic constituents. The antimutagenic effect of the extract and compounds was evaluated using Ames, Vitotox and Comet assays. KEY FINDINGS: Bioassay-guided fractionation of the methanolic leaf extract yielded two antimutagenic compounds identified as (+)-crotepoxide and 5,6-diacetoxy1-benzoyloxymethyl-1,3-cyclohexadiene. Crotepoxide had strong antimutagenicity in the Vitotox assay with an IC50 value of 131 µg/ml. 5,6-Diacetoxy-1-benzoyloxymethyl-1,3-cyclohexadiene showed strong antimutagenic activity in the Ames assay with an IC50 value of 348.9 µg/plate and no antimutagenic activity in the Vitotox test. Furthermore, the compound was able to inhibit, block or prevent biotransformation of aflatoxin B1 by repressing the proteins involved in transcription. CONCLUSIONS: Crotepoxide and 5,6-diacetoxy-1-benzoyloxymethyl-1,3-cyclohexadiene have the potential to mitigate the risks arising from consumption of aflatoxin B1 -contaminated food and feed.

Advantages of a validated UPLC-MS/MS standard addition method for the quantification of A-type dimeric and trimeric proanthocyanidins in cranberry extracts in comparison with well-known quantification methods.

The berries of Vaccinium macrocarpon, cranberry, are widely used for the prevention of urinary tract infections. This species contains A-type proanthocyanidins (PACs), which intervene in the initial phase of the development of urinary tract infections by preventing the adherence of Escherichia coli by their P-type fimbriae to uroepithelial cells. Unfortunately, the existing clinical studies used different cranberry preparations, which were poorly standardized. Because of this, the results were hard to compare, which led sometimes to conflicting results. Currently, PACs are quantified using the rather non-specific spectrophotometric 4-dimethylaminocinnamaldehyde (DMAC) method. In addition, a normal phase HPTLC-densitometric method, a HPLC-UV method and three LC-MS/MS methods for quantification of procyanidin A2 were recently published. All these methods contain some shortcomings and errors. Hence, the development and validation of a fast and sensitive standard addition LC-MS/MS method for the simultaneous quantification of A-type dimers and trimers in a cranberry dry extract was carried out. A linear calibration model could be adopted for dimers and, after logaritmic transformation, for trimers. The maximal interday and interconcentration precision was found to be 4.86% and 4.28% for procyanidin A2, and 5.61% and 7.65% for trimeric PACs, which are all acceptable values for an analytical method using LC-MS/MS. In addition, twelve different cranberry extracts were analyzed by means of the newly validated method and other widely used methods. There appeared to be an enormous variation in dimeric and trimeric PAC content. Comparison of these results with LC-MS/MS analysis without standard addition showed the presence of matrix effects for some of the extracts and proved the necessity of standard addition. A comparison of the well-known and widely used DMAC method, the butanol-HCl assay and this newly developed LC-MS/MS method clearly indicated the need for a reliable method able to quantify A-type PACs, which are considered to be the pharmacologically active constituents of cranberry, since neither the DMAC or butanol-HCl assays are capable of distinguishing between A and B-type PACs and therefore cannot detect adulterations with, for example, extracts with a high B-type PAC content. Hence, the combination of the DMAC method or butanol-HCl assay with this more specific LC-MS/MS assay could overcome these shortcomings.

In Vitro and In Silico Antidiabetic and Antimicrobial Evaluation of Constituents from Kickxia ramosissima (Nanorrhinum ramosissimum).

Background and Aims:Kickxia ramosissima (Wall.) Janch (or Nanorrhinum ramosissimum (Wall.) Betsche is a well-known medicinal plant in Pakistan that is traditionally used in diabetic and inflammatory conditions. Because little information is available on its phytochemical composition, a range of constituents were isolated and evaluated in vitro in assays related to the traditional use. Methods: Dried whole plant material was extracted and chromatographically fractionated. Isolated constituents were evaluated in silico and in vitro in assays related to the traditional use against diabetes (inhibition of α-glucosidase activity; inhibition of advanced glycation endproducts) and in inflammatory conditions (inhibition of AAPH induced linoleic acid peroxidation, inhibition of 15-LOX, antimicrobial activity). Results: Phytochemical analysis of the extracts and fractions led to isolation of 7 compounds, including the iridoids kickxiasine (being a new compound), mussaenosidic acid, mussaenoside and linarioside; the flavonoids pectolinarigenin and pectolinarin; and 4-hydroxy-benzoic acid methyl ester. The iridoids showed weak antiglycation activity. The flavonoids, however, showed interesting results as pectolinarigenin was highly active compared to pectolinarin. In the α-glucosidase inhibition assay, only weak activity was observed for the iridoids. However, the flavonoid pectolinarigenin showed good activity, followed by pectolinarin. In the 15-LOX experiment, moderate inhibition was recorded for most compounds, the iridoids mussaenosidic acid and mussaenoside being the most active. In the AAPH assay, weak or no inhibition was recorded for all compounds. The in silico assays for the α-glucosidase and 15-LOX assays confirmed the results of respective in vitro assays. Pectolinarigenin showed moderate antimicrobial activity against Staphylococcus aureus, Plasmodium falciparum K1, and Trypanosoma cruzi, but it was not cytotoxic on a human MRC-5 cell line. Conclusion: Our findings may in part contribute to explain the traditional use of K. ramosissima.

Selection of chemical markers for the quality control of medicinal plants of the genus Cecropia.

CONTEXT: Several Cecropia (Cecropiaceae) species are traditionally used in Latin America for the treatment of a variety of diseases including diabetes, arterial hypertension, asthma, bronchitis, anxiety, and inflammation. At present, a number of commercial products based on these plants have been introduced into the market with very little information on methods for guaranteeing their quality and safety. OBJECTIVE: This work proposes potential chemical markers for the quality control of the raw materials of Cecropia obtusifolia Bertol., Cecropia peltata L., Cecropia glaziovii Snethl., Cecropia pachystachya Trécul, and Cecropia hololeuca Miq. METHODS: The Herbal Chemical Marker Ranking System (Herb MaRS) developed by the National Institute of Complementary Medicine (NICM) at the University of Western Sydney was used for selecting chemical markers for the quality control of selected medicinal species of Cecropia. This review covers the period from 1982 to 2016. RESULTS: Chlorogenic acid, flavonoidal glycosides (orientin, isoorientin, vitexin, isovitexin, and rutin), catechin, epicatechin, procyanidins (B2, B5, and C1), steroids (ß-sitosterol), and triterpenoids (α-amyrin, pomolic, tormentic and ursolic acids) were selected as chemical markers for the quality control of the leaves. CONCLUSION: It is necessary to establish comprehensive standards for guaranteeing quality, safety and efficacy of herbal drugs. The selection of adequate chemical markers for quality control purposes requires a good knowledge about the chemical composition of medicinal plants and their associated biological properties. To the best of our knowledge this review article is the first to address the identification and quantitative determination of the chemical markers for the genus Cecropia.

In Vitro and In Vivo Study of the Gastrointestinal Absorption and Metabolisation of Hymenocardine, a Cyclopeptide Alkaloid.

Hymenocardine is a cyclopeptide alkaloid present in the root bark of Hymenocardia acida. In traditional African medicine, the leaves and roots of this plant are used to treat malaria, and moderate in vitro antiplasmodial activity has been reported for hymenocardine. However, in view of its peptide-like nature, potential metabolisation after oral ingestion has to be taken into account when considering in vivo experiments. In this study, the stability and small intestinal absorption of hymenocardine was assessed using an in vitro gastrointestinal dialysis model. In addition, potential liver metabolisation was investigated in vitro by incubation with a human S9 fraction. Moreover, hymenocardine was administered to rats per os, and blood and urine samples were collected until 48 and 24 h after oral administration, respectively. All samples resulting from these three experiments were analyzed by LC-MS. Analysis of the dialysate and retentate, obtained from the gastrointestinal dialysis model, indicated that hymenocardine is absorbed unchanged from the gastrointestinal tract, at least in part. After S9 metabolisation, several metabolites of hymenocardine could be identified, the major ones being formed by the reduction and/or the loss of an N-methyl group. The in vivo study confirmed that hymenocardine is absorbed from the gastrointestinal tract unchanged, since it could be identified in both rat plasma and urine, together with hymenocardinol, its reduction product.

Coadministration of a Gloriosa superba extract improves the in vivo antitumoural activity of gemcitabine in a murine pancreatic tumour model.

BACKGROUND: Gloriosa superba L. (glory lily, Colchicaceae) contains colchicine, and related alkaloids such as 3-O-demethylcolchicine and its glycoside colchicoside. Previously the in vivo efficacy of a crude extract and a colchicine-poor / colchicoside-rich extract of G. superba seeds was shown in a murine model of pancreatic adenocarcinoma. HYPOTHESIS/PURPOSE: The efficacy can be improved without obvious signs of toxicity by increasing the treatment dose; the efficacy of gemcitabine can be improved by coadministration of a Gloriosa superba extract. STUDY DESIGN: A survival experiment was carried out in a murine model of pancreatic adenocarcinoma and the semi-long-term toxicity of both G. superba extracts was determined; a combination therapy with gemcitabine was evaluated. METHODS: A crude ethanolic extract (GS) and a colchicine-poor / colchicoside-rich extract (GS2B) were prepared, containing 3.22% colchicine, 2.52% colchicoside and 1.52% 3-O-demethylcolchicine (GS), and 0.07%, 2.26% and 0.46% (m/m) (GS2B). They were evaluated in a murine model of pancreatic adenocarcinoma at a dose of 4.5mg/kg (p.o., daily) total content of colchicine and derivatives during 3 weeks, or at 3.0mg/kg (p.o., daily) combined with gemcitabine (60mg/kg, i.p., 3x/week) during 54 days. RESULTS: A significant effect in tumour growth over time was observed for gemcitabine and the combination therapy compared to the control group. No significant difference was observed for the groups treated with colchicine and both extracts. However, combination therapy was significantly better than the monotherapy with gemcitabine. Moreover, survival analysis showed a significant prolongation of the survival of the groups treated with gemcitabine and the combination therapy. A slight difference in survival was observed between gemcitabine and the combination therapy, the latter one being slightly better. No significant prolongation of survival was observed for the extracts and colchicine compared to the control group. CONCLUSION: Although a relevant tumour growth inhibition and a difference of relative tumour volume compared to the control group were observed on day 11, and a slightly longer survival was noticed for GS2B, the most important conclusion from this study is that the crude G. superba extract (GS) might have an added value combined with gemcitabine in the treatment of pancreatic tumours.

Triterpenoid Saponins from Maesa argentea Leaves.

Within an ongoing research program on saponins with potential antileishmanial activity, four previously undescribed saponins were isolated from Maesa argentea leaves and identified by LC-MS/MS, GC-MS, and 1D and 2D NMR spectroscopy as 3ß-O-{([ß-D-glucopyranosyl-(1 → 2)-α-L-rhamnopyranosyl-(1 → 2)-ß-D-galactopyranosyl-(1 → 3)]-[ß-D-galactopyranosyl-(1 → 2)]-ß-D-glucuronopyranosyl)}-21ß-angeloyloxy-22α-butanoyloxy-13ß,28-oxidoolean-16α,28α-diol (1), 3ß-O-{([ß-D-glucopyranosyl-(1 → 2)-α-L-rhamnopyranosyl-(1 → 2)-ß-D-galactopyranosyl-(1 → 3)]-[ß-D-galactopyranosyl-(1 → 2)]-ß-D-glucuronopyranosyl)}-21ß,22α-angeloyloxy-13ß,28-oxidoolean-16α,28α-diol (2), 3ß-O-{([ß-D-glucopyranosyl-(1 → 2)-α-L-rhamnopyranosyl-(1 → 2)-ß-D-galactopyranosyl-(1 → 3)]-[ß-D-galactopyranosyl-(1 → 2)]-ß-D-glucuronopyranosyl)}-21ß-angeloyloxy-22α-(E)-cinnamoyloxy-13ß,28-oxidoolean-16α,28α-diol (3), and 3ß-O-{([α-L-rhamnopyranosyl-(1 → 2)-ß-D-galactopyranosyl-(1 → 3)]-[ß-D-galactopyranosyl-(1 → 2)]-ß-D-glucuronopyranosyl)}-21ß-angeloyloxy-22α-(E)-cinnamoyloxy-13ß,28-oxidoolean-16α,28α-diol (4). Leaf material was obtained from a germinated seed that was clonally propagated using in vitro tissue culturing. Compounds 1-4 showed structural similarity with maesasaponins and maesabalides reported before from other Maesa spp. All four compounds showed in vitro activity against Plasmodium falciparum K1 and Leishmania infantum at micromolar concentrations. However, the observed inhibitory action must be considered nonspecific since they were also cytotoxic in the same concentration range.

Saponins and Flavonoids from an Infusion of Herniaria hirsuta.

Stone diseases present a major health problem in the Western society, since both urinary and biliary stones occur with a relatively high prevalence of 10-12 % and 10-20 %, respectively, and demonstrate a high recurrence rate. At the moment treatment is mainly based on interventional procedures, or prophylactic and dissolution therapy. However, many of the current drugs cause severe side effects, and therefore, there is an increasing interest in natural medicines. At the moment no registered herbal medicinal products are available for treatment of gallstones. Since an infusion of Herniaria hirsuta L. has a proven efficacy against urolithiasis and cholelithiasis, its phytochemical composition has been investigated. Two previously undescribed triterpene saponins, 28-O-{[ß-D-xylopyranosyl-(1 → 4)-α-L-rhamnopyranosyl-(1 → 2)]-[ß-D-glucopyranosyl-(1-6)]-ß-D-glucopyranosyl}-medicagenic acid and 3-O-[α-L-rhamnopyranosyl-(1 → 3)-ß-D-glucuronopyranosyl]-28-O-{[ß-D-glucopyranosyl-(1 → 3)-ß-D-xylopyranosyl-(1 → 4)]-[ß-D-apiofuranosyl-(1 → 3)]-α-L-rhamnopyranosyl-(1 → 2)-ß-D-fucopyranosyl}-medicagenic acid and three known flavonoids, quercetin-3-O-(2″-O-α-L-rhamnopyranosyl)-ß-D-glucuronopyranoside, rutin, and narcissin (isorhamnetin-3-O-rutinoside), were isolated using flash chromatography and successive semi-preparative HPLC and were well characterized by MS and 1D and 2D NMR spectroscopic techniques. These findings could contribute to the development of a standardized extract that can be used in prophylaxis and treatment of gall and kidney stones.

Bridging the gap between comprehensive extraction protocols in plant metabolomics studies and method validation.

It is vital to pay much attention to the design of extraction methods developed for plant metabolomics, as any non-extracted or converted metabolites will greatly affect the overall quality of the metabolomics study. Method validation is however often omitted in plant metabolome studies, as the well-established methodologies for classical targeted analyses such as recovery optimization cannot be strictly applied. The aim of the present study is to thoroughly evaluate state-of-the-art comprehensive extraction protocols for plant metabolomics with liquid chromatography-photodiode array-accurate mass mass spectrometry (LC-PDA-amMS) by bridging the gap with method validation. Validation of an extraction protocol in untargeted plant metabolomics should ideally be accomplished by validating the protocol for all possible outcomes, i.e. for all secondary metabolites potentially present in the plant. In an effort to approach this ideal validation scenario, two plant matrices were selected based on their wide versatility of phytochemicals: meadowsweet (Filipendula ulmaria) for its polyphenols content, and spicy paprika powder (from the genus Capsicum) for its apolar phytochemicals content (carotenoids, phytosterols, capsaicinoids). These matrices were extracted with comprehensive extraction protocols adapted from literature and analysed with a generic LC-PDA-amMS characterization platform that was previously validated for broad range phytochemical analysis. The performance of the comprehensive sample preparation protocols was assessed based on extraction efficiency, repeatability and intermediate precision and on ionization suppression/enhancement evaluation. The manuscript elaborates on the finding that none of the extraction methods allowed to exhaustively extract the metabolites. Furthermore, it is shown that depending on the extraction conditions enzymatic degradation mechanisms can occur. Investigation of the fractions obtained with the different extraction methods revealed a low resolving power for phytochemicals for all methods. Nevertheless, an overall good repeatability was observed for all extraction methods, which is essential to allow direct comparison between samples. In summary, no single procedure outperforms the others and compromises will have to be made during method selection.

Phytochemical and Pharmacological Investigations on Nymphoides indica Leaf Extracts.

Nymphoides indica (L.) Kuntze (Menyanthaceae) is traditionally used in the Indian subcontinent. However, scientific data reporting its constituents are poor. This study aimed at evaluating its phytochemical constituents and various biological activities. Phytochemical investigations of the extracts and fractions resulted in the isolation of 5 lipophilic compounds, i.e. azelaic (nonanedioic) acid (1) and 4-methyl-heptanedioic acid (3), hexadecanoic (2) and stearic acid (5) and the fatty alcohol hexadecanol (4); 3 seco-iridoids, i.e. 7-epiexaltoside (6), 6″,7″-dihydro-7-epiexaltoside (7) and menthiafolin (8); 3 flavonoids, i.e. 3,7-di-O-methylquercetin-4'-O-ß-glucoside (9), 3-O-methylquercetin-7-O-ß-glucoside (10) and 3,7-di-O-methylquercetin (11); scopoletin (12) and ferulic acid (13); and the monoterpenoids foliamenthoic acid (14) and 6,7-dihydrofoliamenthoic acid methyl ester (15). Compounds 1-5 showed moderate antimicrobial activities, whereas compound 9 presented mild antiprotozoal activities against Trypanosoma brucei (IC50 8 µM), Leishmania infantum (IC50 32 µM) and Trypanosoma cruzi (IC50 30 µM). Antiglycation activity was shown by compounds 7 (IC50 0.36 mM), 10 (IC50 0.42 mM) and 15 (IC50 0.61 mM). Finally α-glucosidase inhibition was shown by compounds 7, 9, 11 and 13-15. It could be concluded that N. indica leaf extracts possess mild to moderate antimicrobial, antiprotozoal, antioxidant and antidiabetic activities. Copyright © 2016 John Wiley & Sons, Ltd.

Efficacy Screening of Gloriosa Superba Extracts in a Murine Pancreatic Cancer Model Using (18)F-FDG PET/CT for Monitoring Treatment Response.

PURPOSE: In vivo efficacy of two herbal extracts of Gloriosa superba L. (Colchicaceae) was investigated in a murine pancreatic tumor model by tumor volume measurements and Positron Emission Tomography (PET) imaging using 2-deoxy-2-[(18)F]fluoro-d-glucose ((18)F-FDG). MATERIALS AND METHODS: A crude extract of G. superba (GS) seeds rich in colchicine and a colchicine-poor extract (GS2B) containing mostly colchicoside as a putative prodrug were prepared. PANC02-bearing C57BL/6 mice were treated with either placebo, gemcitabine, or one of the extracts (three different doses) for 10 days. Tumor volume measurements were performed daily during treatment and additionally (18)F-FDG Positron emission tomography/computed tomography was acquired at baseline and after 7 days of treatment. Ki-67 and cleaved caspase-3 immunostaining was performed on the resected tumors. RESULTS: After 7 days of treatment, a dose-dependent tumor growth inhibition of both extracts was observed with the highest in vivo response at the highest dose of GS and GS2B and gemcitabine. A positive significant correlation was found between Ki-67 scores and relative tumor volumes (RTV), and a negative significant correlation between caspase-3 staining scores and RTV. A decrease in (18)F-FDG uptake was clearly observed in all treatment groups. CONCLUSIONS: The therapeutic efficacy of the two different herbal extracts was demonstrated in an in vivo pancreatic tumor model. (18)F-FDG PET was able to detect an early response as overall lower (18)F-FDG uptake was measured in the treated groups.

A First Step in the Quest for the Active Constituents in Filipendula ulmaria (Meadowsweet): Comprehensive Phytochemical Identification by Liquid Chromatography Coupled to Quadrupole-Orbitrap Mass Spectrometry.

Filipendula ulmaria (meadowsweet) is traditionally used for the treatment of inflammatory diseases and as a diuretic and antirheumatic. Extracts of Filipendulae herba are on the market in the European Union as food supplements. Nevertheless, its active constituents remain to be revealed. During this study, the phytochemical composition of Filipendulae Ulmariae Herba was comprehensively characterised for the first time with two complementary generic ultrahigh-performance liquid chromatography-photodiode array-accurate mass mass spectrometry methods. Selective ion fragmentation experiments with a hybrid quadrupole-orbital trap mass spectrometer significantly contributed to compound identification: a total of 119 compounds were tentatively identified, 69 new to F. ulmaria. A rich diversity of phenolic constituents was detected and only a few non-phenolic phytochemicals were observed. Metabolisation and pharmacological studies should be conducted to investigate which of these constituents or metabolites there of contribute to the activity of F. ulmaria after oral intake.

In vitro and in vivo investigations on the antitumour activity of Chelidonium majus.

BACKGROUND: Chelidonium majus L. (Papaveraceae) (greater celandine) is a medicinal herb that is widely spread in Europe. Antitumoural activity has been reported for C. majus extracts. HYPOTHESIS/PURPOSE: To investigate the antitumour activity of a C. majus extract in vitro and in vivo. STUDY DESIGN: Cytotoxic effects of C. majus extracts were evaluated on human cancer cell lines, i.e. PANC-1 (pancreas cancer), HT-29 (colon cancer), MDA-MB-231 (breast cancer), PC-EM005 and PC-EM002 (primary endometrium cancer cells), and PANC02 (murine pancreatic adenocarcinoma cells). A preliminary in vivo study was performed to evaluate the effect of a defatted C. majus extract and Ukrain(TM) in a highly metastatic murine pancreatic model. METHODS: Chelidonium majus L. herb containing 1.26% (dry weight) of total alkaloids expressed as chelidonine was used to prepare an 80% ethanolic extract (CM2). This crude extract was then defatted with n-hexane, resulting in a defatted C. majus extract (CM2B). Cytotoxic effects of the two extracts (CM2 and CM2B) were evaluated on human and murine cell lines in vitro. CM2B and Ukrain(TM) were evaluated in a highly metastatic murine pancreatic model. RESULTS: Four main benzylisoquinoline alkaloids were identified in CM2B, i.e. chelidonine, sanguinarine, chelerythrine and protopine, using HPLC-UV. CM2 showed a high cytotoxic activity against PANC-1 (IC50, 20.7 µg/ml) and HT-29 (IC50, 20.6 µg/ml), and a moderate cytotoxic activity against MDA-MB-231 (IC50, 73.9 µg/ml). CM2 as well as CM2B showed a moderate to high cytotoxic activity against the PANC02 cell line (IC50, 34.4 and 36.0 µg/ml). Low to almost no cytotoxic effect was observed on primary endometrium cancer cells PC-EM005, PC-EM002 and on normal fibroblast cells 3T3, when treated with CM2B. Significantly less metastases were counted in mice treated with 1.2 mg/kg CM2B, but not with 3.6 mg/kg Ukrain(TM), compared to the control group. The extract, however, did not affect the weight of the primary tumours.

The value of central-African traditional medicine for lead finding: Some case studies.

UNLABELLED: Ethnoparmaological relevance: One of the possible methodologies for the discovery of novel drugs is the screening of selected plant extracts for a broad array of pharmacological activities. MATERIALS AND METHODS: The selection based on enthnomedicinal uses, combined with a follow-up of existing literature on the plants' chemotaxonomic properties, would seem to be the most cost-effective strategy for finding active plant extracts. A bioassay-guided fractionation of the active extracts should subsequently lead to the isolation and identification of the active lead constituent(s). RESULTS AND DISCUSSION: Taking into account the enormous number and the amazing structural diversity of the currently known plant constituents, one might hope that promising model compounds with new structures and/or novel mechanisms of action might be found. In order, however, to optimize such a natural product drug discovery methodology, dereplication and selectivity of activity should be included in the screening system. Dereplication by which known compounds can rapidly be identified from a partially purified mixture prevents a research group from wasting resources by rediscovering known compounds. The use of single-target specific bioassays such as tests on isolated enzymes or on receptor-binding, or multiple target functional bioassays on isolated organs or intact cells must allow at an early stage to isolate compounds with specific pharmacological properties. CONCLUSIONS: In this publication, several examples of bioassay-guided isolation and identification of pharmacologically active lead compounds from plants used in Central-African traditional medicine by our research group will be presented and discussed.

Cholesterol lowering effect in the gall bladder of dogs by a standardized infusion of Herniaria hirsuta L.

ETNOPHARMACOLOGICAL RELEVANCE: Infusions of Herniaria hirsuta L., Herniaria glabra L. and Herniaria fontanesii J.Gay are well known in Moroccon folk medicine for the treatment of biliary dyskinesia, (uro)lithiasis or as a diuretic. Herniariae Herba which can contain H. glabra and H. hirsuta is known in Europe as an urological drug. AIM OF THE STUDY: To investigate the efficacy of a standardized infusion of Herniaria hirsuta against choleltihiasis, and evaluation of its genotoxicity. METHODS AND MATERIALS: An analytical HPLC-UV method to quantify flavonoids and saponins present in the extract of H. hirsuta was developed and validated. An in vivo experiment to evaluate the cholesterol lowering effect of a infusion of H. hirsuta in the gall bladder of dogs was carried out. Dogs were divided into 3 groups i.e. control dogs (CG), dogs treated with ursodeoxycholic acid (UDCA) (2×7.35mg/kg body weight/day) and dogs treated with the standardized infusion (HG) (2×48.5mg/kg body weight/day). Dogs were fed a fatty diet during 120 days after which a diet without additional fat was introduced till day 180. Treatment started 30 days after introduction of the fatty diet and lasted till the end of the experiment. A bile and blood sample of each dog was collected every 30 days, after which the concentration of cholesterol was determined. An Ames test was performed according to the OECD-guidelines. RESULTS: The validated HPLC-UV method showed a linear calibration model and an acceptable precision for the total flavonoid content (total content 4.51%) as well as the total saponin content (12.74%). The in vivo experiments already showed a minor difference for bile cholesterol between CG and HG after 30 days of treatment with the infusion, and the difference was more pronounced after 90 days of treatment. Even 30 days after discontinuation of the cholesterol-rich diet a significant difference remained between CG and HG. There was no statistically significant difference in blood cholesterol. The Ames test showed that the infusion of H. hirsuta could be considered as being free from genotoxic risks. CONCLUSION: A method for the standardization of a infusion of Herniaria hirsuta was developed and validated. Prolonged use of this standardized H. hirsuta extract resulted in a cholesterol-lowering effect in the bile of dogs. Since this pharmacological effect prevents the formation of gallstones and can contribute to solving existing gallstones, a standardized infusion of H. hirsuta may have a positive effect in the treatment of gallstones in human patients.