ABSTRACT
Although some herbal remedies have been used for decades, little is known about the active compounds and the mechanism of action. Many natural products, such as glycosides, can be considered as prodrugs, which become active after biotransformation. To optimize the workflow of in vitro biotransformation followed by automated data analysis, hederacoside C was used as a model compound for saponins. Hederacoside C was subjected to gastrointestinal enzymes and fecal microflora. Samples were analyzed with UHPLC-PDA-HRMS before, during and after in vitro biotransformation, which allowed the monitoring of the relative abundances of the compound and its metabolites. The data-analysis workflow was optimized to render as much information as possible from the longitudinal LCMS data. XCMS was used to convert the raw data into features via peak-picking, followed by grouping, and EDGE was used for the extraction of significant differential profiles. To evaluate if the workflow was suitable for dynamic multiclass metabolomics data, an interactive Shiny web app was developed in R to rate the quality of the resulting features. These ratings were used to train a random forest model for predicting experts response. A performance analysis revealed that the random forest model was capable of correctly predicting the reviewers response in most cases (AUC 0.926 with 10 fold cross validation). The automated data analysis workflow was used for unbiased screening for metabolites and revealed the biotransformation of hederacoside C. As expected, a decrease in relative abundance of hederacoside C was observed over time. Additionally, the relative abundance of metabolites increased, illustrating the biotransformation of hederacoside C, especially in the colon phase, where microbial fermentation takes place. Stepwise progressive elimination of sugar moieties was the major metabolic pathway.
Subject(s)
Herbal Medicine , Metabolic Networks and Pathways , Metabolomics/methods , Oleanolic Acid/analogs & derivatives , Biotransformation , Chromatography, Liquid , Data Analysis , Feces/microbiology , Gastrointestinal Microbiome/physiology , Glycosides/metabolism , Mass Spectrometry , Models, Chemical , Oleanolic Acid/analysis , Oleanolic Acid/metabolism , Saponins/metabolismABSTRACT
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.
Subject(s)
Cecropia Plant/metabolism , Chromatography, High Pressure Liquid/methods , Polyphenols/isolation & purification , Chromatography, Reverse-Phase/methods , Flavonoids/chemistry , Plant Extracts/chemistry , Plant Leaves/chemistry , Polyphenols/analysis , Ultrasonic WavesABSTRACT
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.
Subject(s)
Cecropia Plant/chemistry , Phytochemicals/analysis , Phytochemicals/chemistry , Chromatography, High Pressure Liquid , Cluster Analysis , Molecular Structure , Multivariate Analysis , Panama , Phytochemicals/isolation & purification , Plant Extracts/analysis , Plant Extracts/chemistry , Plant Extracts/isolation & purification , Plant Leaves/chemistryABSTRACT
This work explored the biotechnological potential of the medicinal halophyte Artemisia campestris subsp. maritima (dune wormwood) as a source of health promoting commodities. For that purpose, infusions, decoctions and tinctures were prepared from roots and aerial-organs and evaluated for in vitro antioxidant, anti-diabetic and tyrosinase-inhibitory potential, and also for polyphenolic and mineral contents and toxicity. The dune wormwood extracts had high polyphenolic content and several phenolics were identified by ultra-high performance liquid chromatography-photodiode array-mass-spectrometry (UHPLC-PDA-MS). The main compounds were quinic, chlorogenic and caffeic acids, coumarin sulfates and dicaffeoylquinic acids; several of the identified phytoconstituents are here firstly reported in this A. campestris subspecies. Results obtained with this plant's extracts point to nutritional applications as mineral supplementary source, safe for human consumption, as suggested by the moderate to low toxicity of the extracts towards mammalian cell lines. The dune wormwood extracts had in general high antioxidant activity and also the capacity to inhibit α-glucosidase and tyrosinase. In summary, dune wormwood extracts are a significant source of polyphenolic and mineral constituents, antioxidants and α-glucosidase and tyrosinase inhibitors, and thus, relevant for different commercial segments like the pharmaceutical, cosmetic and/or food industries.
Subject(s)
Antioxidants/analysis , Artemisia/chemistry , Monophenol Monooxygenase/antagonists & inhibitors , Phytochemicals/analysis , Plant Preparations/chemistry , alpha-Glucosidases/metabolism , Antioxidants/pharmacology , Cell Line , Chromatography, High Pressure Liquid , Glycoside Hydrolase Inhibitors/analysis , Glycoside Hydrolase Inhibitors/pharmacology , Health Promotion , Hep G2 Cells , Humans , Phytochemicals/pharmacology , Plant Components, Aerial/chemistry , Plant Roots/chemistry , Tandem Mass Spectrometry , Teas, Herbal/analysisABSTRACT
Several medicinal plants are currently used by the food industry as functional additives, for example botanical extracts in herbal drinks. Moreover, the scientific community has recently begun focusing on halophytes as sources of functional beverages. Helichrysum italicum subsp. picardii (everlasting) is an aromatic halophyte common in southern Europe frequently used as spice and in traditional medicine. In this context, this work explored for the first time H. italicum subsp. picardii as a potential source of innovative herbal beverages with potential health promoting properties. For that purpose, infusions and decoctions were prepared from roots, vegetative aerial-organs (stems and leaves) and flowers and evaluated for in vitro antioxidant and anti-diabetic activities. Samples were also assessed for toxicity in different mammalian cell lines and chemically characterized by spectrophotometric methods and ultra-high performance liquid chromatography-photodiode array-mass-spectrometry (UHPLC-PDA-MS). Results were expressed relating to 'a cup-of-tea' and compared with those obtained with green tea (Camellia sinensis) and rooibos tisane (Aspalathus linearis). Tisanes from the everlasting's above-ground organs, particularly flowers, have high polyphenolic content and several phenolics were identified; the main compounds were chlorogenic and quinic acids, dicaffeoylquinic-acid isomers and gnaphaliin-A. The antioxidant activity of beverages from the everlasting's above-ground organs matched or surpassed that of green tea and rooibos. Its anti-diabetic activity was moderate and toxicity low. Overall, our results suggest that the everlasting is a potential source of innovative and functional herbal beverages.
Subject(s)
Aspalathus , Camellia sinensis , Helichrysum , Animals , Cell Line , Europe , Plant Extracts , Tea , Teas, HerbalABSTRACT
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.
Subject(s)
Cecropia Plant/chemistry , Phytochemicals/standards , Plant Extracts/standards , Quality Control , Animals , Cecropia Plant/classification , Humans , Phytochemicals/isolation & purification , Phytochemicals/therapeutic use , Phytotherapy , Plant Extracts/isolation & purification , Plant Extracts/therapeutic use , Plants, MedicinalABSTRACT
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.
Subject(s)
Alkaloids/metabolism , Embryophyta/chemistry , Gastrointestinal Absorption , Peptides, Cyclic/metabolism , Plant Extracts/metabolism , Alkaloids/blood , Alkaloids/chemistry , Alkaloids/urine , Animals , Chemical Fractionation , Humans , Liver/metabolism , Male , Medicine, African Traditional , Molecular Structure , Peptides, Cyclic/blood , Peptides, Cyclic/chemistry , Peptides, Cyclic/urine , Plant Extracts/blood , Plant Extracts/chemistry , Plant Extracts/urine , Rats , Rats, WistarABSTRACT
Stone diseases present a major health problem in the Western society, since both urinary and biliary stones occur with a relatively high prevalence of 10-12â% and 10-20â%, 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 â 4)-α-L-rhamnopyranosyl-(1 â 2)]-[ß-D-glucopyranosyl-(1-6)]-ß-D-glucopyranosyl}-medicagenic acid and 3-O-[α-L-rhamnopyranosyl-(1 â 3)-ß-D-glucuronopyranosyl]-28-O-{[ß-D-glucopyranosyl-(1 â 3)-ß-D-xylopyranosyl-(1 â 4)]-[ß-D-apiofuranosyl-(1 â 3)]-α-L-rhamnopyranosyl-(1 â 2)-ß-D-fucopyranosyl}-medicagenic acid and three known flavonoids, quercetin-3-O-(2â³-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.
Subject(s)
Caryophyllaceae/chemistry , Flavonoids/chemistry , Saponins/chemistry , Chromatography, High Pressure Liquid , Flavonoids/isolation & purification , Flavonoids/pharmacology , Nuclear Magnetic Resonance, Biomolecular , Saponins/isolation & purification , Saponins/pharmacologyABSTRACT
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.
Subject(s)
Capsicum/chemistry , Filipendula/chemistry , Metabolomics , Plant Extracts/isolation & purification , Capsicum/metabolism , Filipendula/metabolism , Plant Extracts/chemistry , Plant Extracts/metabolismABSTRACT
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.
Subject(s)
Chromatography, Liquid/methods , Filipendula/chemistry , Tandem Mass Spectrometry/methods , Flavonoids/analysis , Flavonoids/chemistry , Phenols/analysis , Phenols/chemistry , Phytochemicals/analysis , Phytochemicals/chemistry , Phytosterols/analysis , Phytosterols/chemistry , Plants, Medicinal/chemistry , Proanthocyanidins/analysis , Proanthocyanidins/chemistryABSTRACT
Alkaline saponification is often used to remove interfering chlorophylls and lipids during carotenoids analysis. However, saponification also hydrolyses esterified carotenoids and is known to induce artifacts. To avoid carotenoid artifact formation during saponification, Larsen and Christensen (2005) developed a gentler and simpler analytical clean-up procedure involving the use of a strong basic resin (Ambersep 900 OH). They hypothesised a saponification mechanism based on their Liquid Chromatography-Photodiode Array (LC-PDA) data. In the present study, we show with LC-PDA-accurate mass-Mass Spectrometry that the main chlorophyll removal mechanism is not based on saponification, apolar adsorption or anion exchange, but most probably an adsorption mechanism caused by H-bonds and dipole-dipole interactions. We showed experimentally that esterified carotenoids and glycerolipids were not removed, indicating a much more selective mechanism than initially hypothesised. This opens new research opportunities towards a much wider scope of applications (e.g. the refinement of oils rich in phytochemical content).
Subject(s)
Chlorophyll/isolation & purification , Plant Extracts/analysis , Adsorption , Carotenoids/analysis , Glycolipids/analysis , Mass Spectrometry , Resins, Plant/chemistryABSTRACT
The aim of the present study was to develop a generic analytical method for the identification and quantitation of apolar plant metabolites in biomass using liquid chromatography-photodiode array-accurate mass mass spectrometry (LC-PDA-amMS). During this study, a single generic sample preparation protocol was applied to extract apolar plant metabolites. Compound identification was performed using a single generic screening method for apolar compounds without the need for dedicated fractionation. Such a generic approach renders vast amounts of information and is virtually limited by only the solubility and detector response of the metabolites of interest. Method validation confirmed that this approach is applicable for quantitative purposes. Furthermore, an identification-quantitation strategy based on amMS and molar extinction coefficients was used for carotenoids, eliminating the need for reference standards for each carotenoid. To challenge the validated method, chili peppers (Capsicum frutescens L.) were analyzed to unravel their complex phytochemical composition (carotenoids, glycolipids, glycerolipids, capsaicinoids, lipid-soluble vitamins).
Subject(s)
Capsicum/chemistry , Fruit/metabolism , Mass Spectrometry/methods , Plant Extracts/chemistry , Capsicum/metabolism , Carotenoids/chemistry , Carotenoids/metabolism , Fruit/chemistry , Plant Extracts/metabolismABSTRACT
Vegetables are a major source of carotenoids and carotenoids are identified as potentially important natural antioxidants that may aid in the prevention of several human chronic degenerative diseases. Characterization of carotenoids in organic biological matrices is a crucial step in any research valorization trajectory. This study reports for the first time the use of high mass resolution and exact mass orbitrap technology for the elucidation of carotenoid fragmentation pathways. This contributes to the generation of new tools for identifying unknown carotenoids based on fragmentation patterns. Two different chromatographic methods making use of different mobile phases resulted in the generation of different ion species because of the large influence of the mobile phase solvent composition on ionization. It was shown that depending on the molecular ion species that are generated (protonated ions or radical molecular ions), different fragments are formed when applying higher energy collisional dissociation. Fragmentation and the abundance of fragments provide valuable structural information on the type of functional groups, the polyene backbone and the location of double bonds in ring structures of carotenoids. Furthermore, coherence between specific substructures in the molecules and characteristic fragmentation patterns was observed allowing the assignment of fragmentation patterns for carotenoid substructures that can theoretically be extrapolated to carotenoids with similar (sub)structures. Differentiation between isomeric carotenoids by compound specific fragments could however not be made for all the isomeric groups under study. As a wide variety of isomeric forms of carotenoids exist in nature, the combination of good chromatographic separation with high resolution mass spectrometry and other complementary qualitative structure elucidation techniques such as a photo diode array detector and/or nuclear magnetic resonance spectroscopy are indispensable for unambiguous identification of unknown carotenoids.