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Nanoparticles are used as carriers for the delivery of drugs and imaging agents. Proteins are safer than synthetic nanocarriers due to their greater biocompatibility and the absence of toxic degradation products. In this context, ferritin has the additional benefit of inherently targeting the membrane receptor transferrin 1, which is overexpressed by most cancer cells. Furthermore, this self-assembling multimeric protein can be loaded with more than 2000 iron atoms, as well as drugs, contrast agents, and other cargos. However, recombinant ferritin currently costs ~3.5 million € g-1 , presumably because the limited number of producers cannot meet demand, making it generally unaffordable as a nanocarrier. Because plants can produce proteins at very-large-scale, we developed a simple, proof-of-concept process for the production of the human ferritin heavy chain by transient expression in Nicotiana benthamiana. We optimized the protein yields by screening different compartments and 5'-untranslated regions in PCPs, and selected the best-performing construct for production in differentiated plants. We then established a rapid and scalable purification protocol by combining pH and heat treatment before extraction, followed by an ultrafiltration/diafiltration size-based separation process. The optimized process achieved ferritin levels of ~40 mg kg-1 fresh biomass although depth filtration limited product recovery to ~7%. The purity of the recombinant product was >90% at costs ~3% of the current sales price. Our method therefore allows the production of affordable ferritin heavy chain as a carrier for therapeutic and diagnostic agents, which is suitable for further stability and functionality testing in vitro and in vivo.

JoVE Biology 20(150), 59933 (2019). doi:10.3791/59933, Published by JoVE, New Delhi

For herbal drugs used for the production of medicinal products, the European Pharmacopoeia has established limits for pesticide residues in Chapter 2.8.13. For pesticide substances not listed in this chapter and whose presence is suspected the limits set by Regulation (EC) No. 396/2005 are applicable. This publication presents an evaluation of a database which includes 3.398.655 sets of data collected between 2011 and 2016 on the occurrence of pesticide residues in 376 herbal drugs. Based on such up-to-date information this contribution aims to support manufacturers of plant-based medicinal products in their individual risk assessment. This may include considerations on which substances are relevant for analysis and which testing frequencies are appropriate. The data show that a number of substances not listed in Ph.Eur. Chapter 2.8.13 can be considered relevant and therefore deserve particular attention within the risk assessment of the manufacturer. Vice versa, some Ph.Eur. substances did not show any positive results in a large number of analyses. For some further substances, the respective Ph.Eur. limits are exceeded only in a few cases. For herbal drugs from organic cultivation a remarkably lower number of positive findings was observed than for conventional cultivation. Regarding positive results for certain substances in organic products, sources other than the application of pesticides, e.g. from environment, packaging material or cross-contamination may be discussed.

Globe artichoke is an ancient herbaceous plant native to the Mediterranean Basin. The edible part of the plant (head) is particularly rich in polyphenols, whose therapeutic properties are well documented. A field experiment was conducted in Sicily (south Italy) to examine the influence of genotype and harvest time on the polyphenol content and profile of different head parts. The concentrations of 19 phenolic compounds were determined by HPLC-DAD-ESI/MS n analysis. It was observed that individual phenolic substances were preferentially accumulated in specific head parts and genotypes. Apigenin 7- O -glucuronide was found to be the major flavonoid, with 6298 mg kg −1 DM in ‘Romanesco clone C3’ receptacle, whereas chlorogenic acid represented the main caffeoylquinic acid, reaching 14841 mg kg −1 DM in the inner bracts of ‘Violetto di Sicilia’. Our findings prove also the influence of climatic conditions on the phenolic profile and thus suggest giving specific consideration to harvest time.

Since durum wheat is ∼20% more expensive than common wheat and considered of superior quality for the manufacture of pasta products, efficient methods for the detection of accidental or intentional admixtures of common wheat to durum wheat products are required. This paper describes a novel approach for the detection and quantification of whole grain common wheat adulteration in whole grain durum flour and dried pasta. We found that differences in the C17:0 to C21:0 alkylresorcinol homologue ratios between the two cereal species may serve as a suitable tool for whole grain durum product authentification. To detect and estimate adulteration, the C17:0/C21:0 ratios of flour and pasta admixtures with added whole grain flour of common wheat were analysed. A linear relationship between C17:0/C21:0 ratios and level of admixture in pasta samples showed that adulteration can be estimated within the range of 5–100% of admixture. Furthermore, di- and triunsaturated as well as oxygenated alk(en)ylresorcinols are reported to occur in Triticum durum Desf. for the first time.

Gallotannins were extracted from mango ( Mangifera indica L.) kernels with aqueous acetone (80%, v/v) and purified using liquid-liquid extraction and two-step low-pressure liquid chromatography (LPLC) on Sephadex LH-20. Analytical high-performance liquid chromatography and mass spectrometry confirmed the presence of hydrolyzable tannins with a degree of galloylation ranging from 4 to 9 and additionally revealed the presence of deca-, undeca-, and dodeca-O-galloylglucose. Further purification using two-step semipreparative HPLC resulted in three pure hydrolyzable tannins, penta-, hexa-, and hepta-O-galloylglucose, with antibacterial activity, as evidenced from the agar spot and critical dilution assays. Although the growth of lactic acid bacteria was not inhibited, the proliferation of Gram-positive food spoilage bacteria was prevented and the growth of Gram-negative Escherichia coli was reduced. Because bacterial growth could be restored by the addition of iron to the medium, this study strongly supports the view that the inhibitory effects of hydrolyzable tannins are due to their iron-complexing properties.

Despite a number of serious case reports of mango dermatitis, no attempts at the identification and quantification of allergenic 5-alk(en)ylresorcinols in mango fruits have so far been made. Therefore, total alk(en)ylresorcinol content and relative homologue composition in 13 mango peel samples and 7 samples of mango pulp were determined by HPLC and LC-MS/MS analyses. Furthermore, mango puree and nectar prepared on pilot plant scale were also analyzed and compared with commercially available thermally preserved products. Depending on cultivar, alk(en)ylresorcinol contents ranged from 79.3 to 1850.5 mg/kg of dry matter (DM) in mango peels and from 4.9 to 187.3 mg/kg of DM in samples of mango pulp. The profile of alk(en)ylresorcinols was found to be highly characteristic, with an average homologue composition of C15:0 (6.1%), C15:1 (1.7%), C17:0 (1.1%), C17:1 (52.5%), C17:2 (33.4%), C17:3 (2.4%), C19:1 (2.1%), and C19:2 (0.8%). Mango puree samples prepared from peeled and unpeeled fruits revealed contents of 3.8 and 12.3 mg/kg of fresh weight, respectively. Content and homologue composition were not significantly affected during puree processing and thermal preservation. In nectar samples prepared from peeled and unpeeled fruits, contents of 1.4 and 4.6 mg/L, respectively, were found.

5-Alkyl- and 5-alkenylresorcinols, as well as their hydroxylated derivatives, were extracted from mango (Mangiferaindica L.) peels, purified on polyamide and characterized by high-performance liquid chromatography/atmospheric pressure chemical ionization mass spectrometry (HPLC/APcI-MS) for the first time. Among the 15 compounds analyzed, 3 major and 12 minor C15-, C17-, and C19-substituted resorcinols and related analogues, showing varying degrees of unsaturation, were characterized by their specific fragmentation patterns in collision-induced dissociation experiments. This marks the first report on the occurrence of mono-, di-, and triunsaturated C15-homologues, saturated and triunsaturated C17-homologues, and mono- and diunsaturated C19-homologues in mango peels. Additionally, several hydroxylated C15- and C17-homologues, also not yet described in mango, and a C14-monoene, unique because of its even-numbered side chain, were tentatively identified on the basis of their fragmentation patterns. The results obtained in the present study indicate that HPLC-DAD-APcI-MSn, combined with the newly developed solid-phase extraction, is a powerful tool for the analysis of alk(en)ylresorcinols and could therefore be used for their determination in various matrices. Copyright © 2007 John Wiley & Sons, Ltd.

Two different options for the combined recovery of pectin and phenolic compounds from mango peels, a byproduct of industrial mango processing, were developed. After extraction of dried mango peels with diluted sulfuric acid, the phenolic compounds were adsorbed using a styrene–divinylbenzene copolymerisate resin, and pectin was obtained from the effluent by precipitation with ethanol. Phenolic compounds were recovered from the resin with methanol and the eluate was lyophilized (Process I). Alternatively, the pectin was precipitated by adding the crude extract to ethanol. After removal of the organic solvent, the phenolic compounds were obtained from the aqueous phase of the precipitation bath using the adsorbent resin as described before (Process II). While in total, 129.4 mg/g polyphenols were detected in the lyophilizate obtained from Process I, only 71.0 mg/g dm could be recoverd from Process II. The profiles of the polyphenols were almost identical, revealing that during pectin precipitation preferential adsorption of polyphenolic compounds to the pectin may be excluded. Besides the characterization of the pectins and the phenolic compounds, investigations into the influence of the drying temperature on the polyphenolic content of the peels were carried out, indicating a significant loss of flavonol glycosides depending on heat exposure. On the other hand, some xanthone glycosides were formed during the drying process. Furthermore, antioxidative capacities of the lyophilized eluates were investigated using the DPPH, TEAC and FRAP assays. The antioxidative capacity of the extracts exceeded that of mangiferin and quercetin 3-O-glucoside, respectively, thus demonstrating mango peels to be a suitable source of health-beneficial compounds. The lyophilizates obtained from Process I showed higher antioxidative capacities in all three assays. These findings indicate a correlation between the amount of phenolic compounds and the antioxidative capacity. Industrial relevance Byproducts of mango processing amount to 35–60% of the total fruit weight. Their complete exploitation for further product recovery is a promising measure from both an environmental and economic point of view. In our previous study mango peels were found to be a rich source of pectin, with a high degree of esterification and phenolic compounds, like flavonol O- and xanthone C-glycosides. Therefore, two alternative processes for the combined recovery of pectin and polyphenols, which can easily be integrated in an existing pectin production process, were developed in the present study.

Mercurialis annua and M. perennis are medicinal plants used in complementary medicine. In the present work, analytical methods to allow a chemotaxonomic differentiation of M. annua and M. perennis by means of chemical marker compounds were established. In addition to previously published compounds, the exclusive presence of pyridine-3-carbonitrile and nicotinamide in CH(2) Cl(2) extracts obtained from the herbal parts of M. annua was demonstrated by GC/MS. Notably, pyridine-3-carbonitrile was identified for the first time as a natural product. Further chromatographic separation of the CH(2) Cl(2) extracts via polyamide yielded a MeOH fraction exhibiting a broad spectrum of side-chain saturated n-alkylresorcinols. While the n-alkylresorcinol pattern was similar for both plant species, some specific differences were observed for particular n-alkylresorcinol homologs. Finally, the investigation of H(2) O extracts by LC/MS/MS revealed the presence of depside constituents. Whereas, in M. perennis, a mixture of mercurialis acid (=(2R)-[(E)-caffeoyl]-2-oxoglutarate) and phaselic acid (=(E)-caffeoyl-2-malate) could be detected, in M. annua solely phaselic acid was found. By comparison with synthesized enantiomerically pure (2R)- and (2S)-phaselic acids, the configuration of the depside could be determined as (2S) in M. annua and as (2R) in M. perennis.

Investigation of the dichloromethane extracts from herbal and root parts of Mercurialis perennis L. afforded a mixture of 11 homologous n-alkylresorcinols (ARs) with saturated odd-numbered alkyl side chains (C15:0-C27:0). In addition to three predominant ARs (C19:0, C21:0 and C23:0), a number of minor ARs were identified by use of LC-MS/MS and GC-MS techniques. Among the compounds detected, four uncommon ARs with evennumbered alkyl side chain lengths were also determined. The overall AR concentration in herbal parts was 7 to 9 times higher compared to that of the roots. The results presented may open a new view on the phytochemistry and pharmacognosy of M. perennis and other members of the Euphorbiaceae family.

5-Alk(en)ylresorcinols in rye, wheat, spelt, and barley have been characterized by high-performance liquid chromatography coupled to atmospheric pressure chemical ionization multistage mass spectrometry (HPLC-APcI-MS n ) for the first time. Among the 29 compounds analysed, several major and minor C15, C17, C19, C21, C23, and C25-substituted resorcinols with saturated, monoenoic, dienoic, and/or oxygenated side-chains were characterized by their specific fragmentation patterns in collision-induced dissociation experiments. Additionally, a C27:0 homologue, which has probably been overlooked in previous studies based on HPLC alone, was detected in all cereals analysed. Furthermore, we provide tentative evidence for the occurrence of alkylresorcinols with triolefinic side-chains, which have, to our knowledge, so far not been reported in any cereal species.

Bioassay directed extraction and purification of mango peels revealed the 5-(11′ Z -heptadecenyl)-resorcinol ( 1 ) and the known 5-(8′ Z ,11′ Z -heptadecadienyl)-resorcinol ( 2 ) previously not described in Mangifera indica L. The structures of both compounds were determined by extensive 1D and 2D NMR studies and MS. Both compounds exhibited potent cyclooxygenase (COX)-1 and COX-2 inhibitory activity with IC 50 values ranging from 1.9 ( 2 ) to 3.5 μM ( 1 ) and from 3.5 ( 2 ) to 4.4 ( 1 ) μM, respectively, coming close to the IC 50 values of reference drugs. 5-Lipoxygenase (5-LOX) catalyzed leukotriene formation was only slightly inhibited. Structure–activity studies by referring to synthetic saturated homologues indicated that the degree of unsaturation in the alkyl chain plays a key role for COX inhibitory activity, whereas the influence of chain length was less significant.